Title Pathogenesis of Chlamydial Infections( 本文（FULLTEXT ...repository.lib.gifu-u.ac.jp/bitstream/20.500.12099/21409/...conjunctivitis in cats, particularly kittens (192). Studies

Title Pathogenesis of Chlamydial Infections( 本文（FULLTEXT) )

Author(s) RAJESH, CHAHOTA

Report No.(DoctoralDegree) 博士(獣医学) 甲第226号

Issue Date 2007-03-13

Type 博士論文

Version author

URL http://hdl.handle.net/20.500.12099/21409

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Pathogenesis of Chlamydial Infections

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2006

The United Graduate School of Veterinary Sciences, Gifu University,

(Gifu University)

RAJESH CHAHOTA

Pathogenesis of Chlamydial Infections

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RAJESH CHAHOTA

i

CONTENTS

PREFACE……………………………………………………………………… 1

PART I

Molecular Epidemiology, Genetic Diversity, Phylogeny and Virulence Analysis

of Chlamydophila psittaci

CHAPTER I: Study of molecular epizootiology of Chlamydophila psittaci among

captive and feral avian species on the basis of VD2 region of ompA gene

Introduction……………………………………………………………… 7

Materials and Methods…………………………………………………... 9

Results…………………………………………………………………… 16

Discussion……………………………………………………………….. 31

Summary……………………………………………………………….... 35

CHAPTER II: Analysis of genetic diversity and molecular phylogeny of the

Chlamydophila psittaci strains prevalent among avian fauna and those associated with

human psittacosis

Introduction……………………………………………………………… 36


Results…………………………………………………………………… 42

Discussion……………………………………………………………….. 55

Summary………………………………………………………………… 59

CHAPTER III: Examination of virulence patterns of the Chlamydophila psittaci strains

predominantly associated with avian chlamydiosis and human psittacosis using BALB/c

mice

Introduction……………………………………………………………… 60


Results…………………………………………………………………… 66

Discussion……………………………………………………………….. 74

Summary………………………………………………………………… 76

ii

PART II

Investigation of the Emergence of Chlamydial Infection in Animals and Human

Disease Conditions

CHAPTER IV: Involvement of multiple Chlamydia suis genotypes in porcine

conjunctivitis in commercial farms of Japan

Introduction……………………………………………………………… 79


Results…………………………………………………………………… 86

Discussion……………………………………………………………….. 92

Summary………………………………………………………………… 98

CHAPTER V: Molecular characterization of the Chlamydophila caviae strain OK135,

isolated from human genital tract infection, by analysis of some structural and

functional genes

Introduction……………………………………………………………. 99

Materials and Methods………………………………………………… 100

Results…………………………………………………………………. 103

Discussion……………………………………………………………... 108

Summary………………………………………………………………. 110

CONCLUSION………………………………………………………………… 112

REFERENCES…………………………………………………………………. 116

ACKNOWLEDGMENTS……………………………………………………… 140

1

Chlamydiae are the obligate intracellular bacterial pathogens of animals and human

beings (185, 188). They replicate within the cytoplasm of host cells, forming characteristic

intracellular inclusion bodies. The chlamydiae undergo a unique biphasic developmental

cycle characterized by two morphologically distinct forms, the elementary body (EB) and

the reticulate body (RB). EBs are small (0.2–0.3 µm in diameter) infectious forms that are

either endocytosed or phagocytosed by host cells. The EB within an intracytoplasmic

inclusion, transforms into the larger (0.5–1.6 µm in diameter) intracellular non-infectious,

metabolically active, RB. The RB multiplies by binary fission and fills the inclusion,

which expands in size. RBs re-condense back into EBs via intermediate bodies towards the

end of the cycle (24–48 hr, depending on species and strains) and are then released from the

host cell by lysis or exocytosis to initiate another cycle of infection (75, 119, 131, 216).

The EB, in contrast to the RB, is structurally rigid, resulting from the formation of

extensive disulphide linkages between various cysteine-rich proteins in or associated with the

outer membrane. The chlamydial outer membrane consists of lipopolysaccharide (LPS),

polymorphic outer membrane proteins (POMPs), peptidoglycan and cysteine-rich proteins

like major outer membrane protein (MOMP), OmcA and OmcB. The most important of

these is the MOMP, which makes up 60% of the weight of the outer membrane. MOMP is

about 40- to 45-Kda in mass and contains serovar- and species-specific epitopes. MOMP

acts as porins, channels to allow exchange of molecules including nutrients. The high

rigidity results in EBs being resistant to both chemical and physical factors and therefore,

adapted for prolonged extracellular survival, an important factor in terms of chlamydial

pathogenesis and control or treatment of chlamydial infections (75, 176).

Earlier all chlamydiae were placed in order Chlamydiales, family Chlamydiaceae

and one genus Chlamydia that included 4 species: C. trachomatis, C. psittaci, C.

PREFACE

2

pneumoniae and C. pecorum (52, 64, 132). Following reclassification of the order

Chlamydiales in 1999, the family Chlamydiacaeae is now divided into two genera,

Chlamydia and Chlamydophila (43). The genus Chlamydia comprises of Chlamydia

trachomatis, Chlamydia suis and Chlamydia muridarum species. The genus

Chlamydophila contains the species Chlamydophila pneumoniae, Chlamydophila psittaci,

Chlamydophila abortus, Chlamydophila pecorum, Chlamydophila felis and Chlamydophila

caviae.

Chlamydiae are responsible for a diverse range of diseases in birds and mammals

including humans. Among human chlamydial infections, C. trachomatis causes blindness

(trachoma), sexually transmitted disease and infertility. C. pneumoniae is a cause of acute

respiratory infection, coronary heart disease and chronic inflammatory lung diseases viz.

chronic obstructive pulmonary disease and asthma (71, 105, 177).

Animal chlamydial infections are caused by many chlamydial species. In genus

Chlamydia, C. suis is associated with either asymptomatic infections or cause

conjunctivitis, enteritis, pneumonia and genital tract infections in domesticated and wild

porcine (99, 165, 183). C. muridarum causes either no disease or asymptomatic

pneumonitis among mice and hamsters (40, 48). In genus Chlamydophila, C. pecorum is

associated with chronic conditions viz. pneumonia, polyarthritis, conjunctivitis, enteritis,

encephalomyelitis, metritis and mastitis among ruminants, swine and marsupials (40, 52).

C. felis is responsible for “feline chlamydiosis”, which is characterized by rhinitis and

conjunctivitis in cats, particularly kittens (192). Studies conducted in Japan have shown C.

felis to be more widespread in stray cats than in pet cats (26, 57, 222). C. abortus is a cause

of abortion in sheep, goats and responsible for reproductive failure in bovine, equine and

porcine (40, 113, 160). C. psittaci causes “avian chlamydiosis” among many avian species

(reported 460 avian species) and can also infect mammalian species (8, 94, 210). Avian

chlamydiosis is characterized by diarrhea, anorexia, respiratory distress, sinusitis, rhinitis

3

and conjunctivitis (8, 188). C. caviae is a host specific pathogen of guinea pigs and causes

either self-limiting or asymptomatic inclusion conjunctivitis among young (4-8 weeks old)

guinea pigs (63, 101, 135). C. pneumoniae is also reported from marsupials and equines

(62, 218).

Animal chlamydial infections also have public health hazard significance. Among

all chlamydial species infecting animals, C. psittaci, C. abortus and C. felis are potential

zoonotic pathogens (113). C. abortus can cause severe, life-threatening disease in pregnant

women. Infection in women during pregnancy can result in spontaneous abortion or

stillbirths, which are typically preceded by several days of acute influenza-like illness, as well

as renal failure, hepatic dysfunction, disseminated intravascular coagulation, and possibly

death (25, 87). Cases of C. felis infection associated with conjunctivitis and atypical

pneumonia in humans have been reported over the years (19, 113), but it is only recently

that compelling evidence of transmission from cats to humans has been provided by way

of molecular classification techniques (74). In Japan serological evidence of human C. felis

infection has been reported (222).

The zoonotic infection by C. psittaci, is particularly a significant occupational health

hazard to the workers in poultry industry, aviaries, zoos and also pet shop owners, farmers

and veterinarians. Infection in humans can vary in severity but is usually associated with

respiratory disease. But multiple organs may become infected resulting in endocarditis,

myocarditis, hepatitis, encephalitis or meningitis and result in deaths (113, 181, 210). The

financial losses resulting from this disease, particularly those incurred in the poultry and pet

industries, combined with the fact that this is the most common animal chlamydiosis

transmissible to man, highlights the economic importance and public health significance of

avian chlamydiosis. The incidences of zoonotic infection by C. caviae, C. pecorum, C. suis

and C. muridarum are unknown (113).

Defining the distribution of natural chlamydial infections in animals and human and

its relationship to pathogenesis provides the fundamental basis for management and control

4

of chlamydial diseases. The final outcome of chlamydial infection in animals and human

depends on large numbers of host and organism specific factors (217). Genetic and

biological differences among various chlamydial species/strains are well known. Different

pathobiotypes of C. trachomatis strains have been recognized, which include trachoma,

urogenital and lymphogranuloma venereum (LGV) biovars. These biovars varies in tissue

tropism and host specific cellular response which indicates variation in disease causation

mechanism (128, 137, 214). In C. pneumoniae also, human, equine and koala biovars are

known. The virulence and genetic distinction of C. abortus strains causing abortion in

ewes from non abortifacient strains has also been established (38, 159, 162). However, in

C. psittaci no such relationship has been explored among various genetically diverse

strains even though it has been detected from diverse host rage and disease conditions. On

the other hand, some studies have found varied virulence of C. psittaci strain to avian

species (212). Therefore, detailed molecular epidemiological studies of animal chlamydial

species by screening variety of host species and disease conditions is important to identify

association of particular species/strain with specific disease syndrome.

The diagnosis of chlamydial infections and analysis of chlamydial diversity are

done by isolation, serological and biotyping studies (175, 184, 220). But these procedures

are time consuming and high technical skills are required for testing samples. Therefore,

the recent epidemiological studies are mostly contemplated by detecting species/strain

specific gene (DNA) by nucleic acid amplification using polymerase chain reaction (PCR).

By this technique even a very small amount of EBs can be detected. The genetic diversity

among the prevalent chlamydial species/strains can be studied by sequencing and

genetically analyzing the amplified DNA fragment (59, 80, 83, 96, 97, 148).

The differentiation at the species level among various chlamydiae is usually done

by genetic analysis of highly conserved genes like 16S ribosomal RNA, 16S-23S

5

ribosomal RNA intergenic spacer region, rnpB and groEL-1 genes (24, 41, 54, 79, 149,

193, 205). However, for the identification at strain level, ompA gene coding MOMP has

been targeted in many studies (38, 47, 50, 60, 173, 191).

For identification and differentiation of C. psittaci isolates especially of avian

origin, ompA gene was analyzed by many researchers (50, 98, 174, 191, 207). Depending

on serovar specific monoclonal antibody typing, 8 serotypes named A to H have been

identified in C. psittaci (4, 56, 206, 207). Based on AluI restriction fragment length

polymorphism (RFLP) pattern of ompA gene, C. psittaci strains are also divided into 7

genotypes from A to F and E/B (60, 174, 207), but serotyping and RFLP studies do not

reflect the actual genetic diversity (60).

Therefore, the detailed investigations about natural distribution, molecular

identification, genetic diversity and virulence of various chlamydial species/strains of

animal origin are necessary to understand disease pathogenesis. Hence, to elucidate some

of these questions the present study was undertaken with the following objectives:

1) Study of molecular epizootiology of Chlamydophila psittaci among captive and feral

avian species on the basis of VD2 region of ompA gene.

2) Analysis of genetic diversity and molecular phylogeny of Chlamydophila psittaci

strains prevalent among avian fauna and those associated with human psittacosis.

3) Examination of virulence patterns of Chlamydophila psittaci strains predominantly

associated with avian chlamydiosis and human psittacosis using BALB/c mice.

4) Investigation of the emergence of chlamydial infection among animals and human

disease conditions.

These findings will help to identify predominant/emergent chlamydial genotypes

prevalent among domesticated and wild fauna, with/without disease symptoms and also

those with high zoonotic potential for better understanding of disease pathogenesis.

6

PART I

Molecular Epidemiology, Genetic Diversity, Phylogeny and Virulence

Analysis of Chlamydophila psittaci

7

f

Study of molecular epizootiology of Chlamydophila psittaci among captive

and feral avian species on the basis of VD2 region of ompA gene

INTRODUCTION

Chlamydiae are obligate intracellular pathogens and widely prevalent among avian

species, animals and human beings (7, 51, 132, 188). According to the recent classification

based upon 16S rRNA gene sequences, all the chlamydiae are divided into four families

(43). Chlamydophila species belonging to the family Chlamydiaceae are often involved in

the infection of avian species and domestic animals (43, 53).

The avian chlamydiosis is caused by Chlamydophila psittaci. The disease is

characterized by clinical and/or subclinical infection. The clinical signs vary in severity

and disease is usually systemic with up to 30% mortality (8, 188). C. psittaci has been

reported in more than 400 avian species and occasionally in non-avian hosts (8, 94, 143,

182, 209). C. psittaci is usually transmitted horizontally to in-contact animals and human

beings by diseased or subclinically infected birds (35, 112, 113, 115, 181). People are

frequently exposed to diverse type of domestic and/or feral avian fauna in daily life either

incidentally or occupationally. Furthermore, the feral birds congregated in urban public

habitations also contaminate the environment with C. psittaci laced aerosols and droppings

(30, 55, 70, 202). In Japan, 45 cases of human psittacosis in 2002, 44 in 2003 and 36 in

2004 were reported (136) and in USA 935 cases were reported from 1988 to 2003 (181).

Many more cases may have occurred but are neither correctly diagnosed nor reported due

to confusing symptoms.

CHAPTER-I

8

Immunodiagnostic and biotyping studies (31, 184, 220) were reported to identify

various avian species harboring C. psittaci and to detect chlamydial diversity. Depending

on serovar specific monoclonal antibody typing, 8 serotypes named A to H have been

identified in C. psittaci (4, 56, 206, 207). In the last decade, for identification and

differentiation of C. psittaci isolates especially of avian origin, ompA gene coding major

outer membrane protein (MOMP) was analyzed by many researchers (50, 98, 174, 191,

207). On the basis of AluI restriction fragment length polymorphism (RFLP) pattern of

ompA gene, C. psittaci strains are also divided into 7 genotypes from A to F and E/B (60,

174, 207), but serotyping and RFLP studies do not reflect the actual genetic diversity (60).

The ompA gene consists of genetically conserved fragments called conserved/constant

domains (CD) flanking 4 genetically variable domains (VD). The VDs of ompA gene are

reported to have species/strain specific sequences and thus can be analyzed for

identification of chlamydial species/strains and also to study the genetic diversity directly

from field samples after PCR amplification (12, 86, 96, 148, 225).

The detailed epizootiological study of avian chlamydiosis is essential to know the

prevalence of different chlamydial strains/species among various avian species and to

ascertain the possibility of health hazard risk to in-contact animals and human beings.

Moreover, limited information is available in literature on the current molecular

epizootiology and genetic diversity of C. psittaci after reclassification of avian and

mammalian isolates of the previous Chlamydia psittaci group (43). Therefore, the present

research was planned to investigate the molecular epizootiology and genetic diversity

among C. psittaci strains and/or other chlamydial species prevalent in diverse avian fauna.

It appears that various genetically diverse chlamydial species and strains may cause avian

chlamydiosis but some strains of C. psittaci are highly prevalent and are frequently

associated with clinical/subclinical infections.

9

MATERIALS AND METHODS

Samples examined: A total of 1,147 samples from 11 avian orders were collected

from 4 avian pet shops, 3 bird sanctuaries/wild animal rehabilitation centers, 3 bird

parks/zoos and 14 veterinary hospitals located in 11 prefectures of Japan from January

2003 to December 2004. The collected samples included the cloacal swabs or freshly

voided feces and/or whole blood in heparin and/or pieces of visceral organs (1-2 g)

including lung, liver, spleen, heart and posterior intestinal loop from dead birds. The avian

fauna screened in the study is shown in Table 1. The distribution of tested samples

according to clinical history and sources are shown in Table 2. In total, 11 avian orders

include 28 genera and 81 species from psittacine birds and 25 genera comprising of 32

species from non-psittacine birds.

DNA extraction: SepaGene DNA extraction Kit (Sanko Junyaku, Tokyo, Japan)

was used to extract DNA from samples according to the manufacturer’s instructions. In

brief, about 200 mg of fecal/tissue material or 50 µl of blood or cloacal swabs suspended

in 700 µl of phosphate-buffered saline (PBS), pH 7.4 were processed for DNA extraction.

DNA was finally dissolved in 30 µl Tris-EDTA (TE) buffer, pH 7.4 (100 mM Tris-HCl,

pH 7.4 and 10 mM EDTA, pH 8.0) and stored at –30°C. DNA extracted from purified

elementary bodies of GPIC strain of Chlamydophila caviae (ATCC VR-813) was used as a

positive control in the test.

Nested PCR: Two sets of consensus oligonucleotide primers based on ompA gene

were used in a two-steps procedure. An outer pair of primers CMGP-1F (5'-

CCTTGTGATCCTTGCGCTACTTG-3'; nucleotide (nt) 138 to 160 in ompA gene

sequence of 6BC strain of C. psittaci with accession number X56980) and CMGP-1R (5'-

GTGAGCAGCTCTTTCGTTGAT-3'; nt 1184 to 1164) and an inner pair of primers

Table 1. Details of the avian species screened in the study.

Order Family Genus (sample no.) Avian species and number of samples testeda

Psittaciformes Cacatuidae Cacatua (98) C. alba (Umbrella or White-crested Cockatoo)-27; C. galerita (Sulphur-crested Cockatoo)-3; C. triton (Tritone Cockatoo)-3; C. tenuirostrius

(Long-billed Corella)-1; C. sulphurea (Yellow-crested Cockatoo)-26; C. sanguinea (Little Cockatoo)-1; C. leadbeateri (Pink Cockatoo)-2;

C. sulphurea citrinocristana (Citron-crested Cockatoo)-17; C. moluccensis (Moluccan or Salmon-crested Cockatoo)-18

Eolophus (9) E. roseicapillus (Roseate Cockatoo or Galah)-9

Nymphicus (77) N. hollandicus (Cockatiel)-77

Psittacidae Agapornis (11) A. roreicollis (Peached-faced Lovebird)-10; A. lilianae (Lilian Lovebird)-1

Amazona (13) A. aestiva (Blue-fronted Amazon)-6; A. aestiva xanthopteryx (a type of Blue-fronted Amazon)-1; A. ochrocephala (Yellow-crowned Amazon)-3;

A. auropalliata (Yellow-naped Amazon)-1; A. xanthops (Yellow-faced Amazon)-1; A. farinosa (Mealy Amazon)-1

Ara (55) A. nobilis (Red-shouldered Macaw)-2; A. chloroptera (Green-winged Macaw)-10; A. ararauna (Blue-and-Yellow Macaw)-11; A. severa

(Chestnut-fronted Macaw)-7; A. auricollis (Yellow-collard Macaw)-23; Ara sp. (Harlequin Macaw)-1; A. macao (Scarlet Macaw)-1

Aratinga (44) A. aurea (Peach-fronted Conure)-3; A. wagleri (Scarlet or Red-fronted Conure)-2; A. jandaya (Jandaya Conure)-6; A. solstitialis (Sun Conure)-9;

A. erythrogenys (Red-masked Conure)-5; A. weddellii (Dusky-headed Conure)-5; A. acuticaudata (Blue-crowned Conure)-7; A. pertinax

(St. Thomas Conure)-1; A. guarouba (Golden Conure)-2; A. rubritorquis (Red-throated Conure)-4

Bolborhynchus (1) B. lineola (Barred Parakeet )-1

Brotogeris (2) B. chrysopterus (Golden-winged Parakeet )-2

Cyanoliseus (1) C. patagonus (Patagonia Conure)-1

Eclectus (10) E. roratus (Eclectus Parrot)-10

Eos (3) E. bornea (Red Lory)-1; E. reticulata (Blue-streaked Lory)-2

Forpus (1) F. coelestis (Pacific Parrotlet)-1

Lorius (11) L. lory (Black-capped Lory)-5; L. garrulus (Chattering Lory)-1; L. chlorocerus (Yellow-bibbed Lory)-5

Melopsittacus (59) M. undulatus (Budgeriger)-59

Myiopsitta (1) M. monachus (Quaker or Monk Parrot)-1

Neophema (2) N. bourkii (Bourke's Parrot)-2

Pionites (14) P. melanocephala (Black-headed Caique)-6; P. leucogaster (White-bellied Caique )-8

Pionus (12) P. senilis (White-crowned Parrot)-4; P. fuscus (Dusky Parrot)-1; P. menstruus (Blue-headed Parrot)-4; P. chalcopterus (Bronze-winged Parrot)-3

Platycerus (1) P. elegans (Crimson Rosella)-1

Poicephalus (121) P. cryptoxanthus (Brown-headed Parrot)-26; P. meyeri (Meyer's Parrot/Broun Parrot)-19; P. rueppellii (Rüppell's parrot)-7; P. rufiventris

(Red-bellied Parrot)-4; P. senegalus (Senegal Parrot)-42; P. gulielmi (Red-crowned/Jardine's Parrot)-23

Polytelis (3) P. anthopeplus (Regent Parrot)-2; P. alexandrae (Alexandra's Parrot)-1

Pseudeos (7) P. fuscata (Dusky Lory)-7

Psittacula (12) P. krameri manillensis (Indian Ring-neck Parakeet)-2; P. eupatria (Alexandrine Parakeet)-4; P. derbiana (Derbyan Parakeet)-3; P. cyanocephala

(Plum-headed Parakeet)-3

Psittacus (163) P. erithacus (African Grey Parrot)-154; P. erithacus timneh (Timneh Grey Parrot)-9

Pteroglossus (2) P. aracari (Black-necked Aracari)-2

Pyrrhura (21) P. molinae (Green-cheecked Conure)-4; P. egregia (Fiery-shouldered Conure)-3; P. rupicola (Rock or Black-capped Conure)-2; P. frontalis

(Red-bellied Conure)-3; P. rhodocephala (Rose-headed Conure)-1; P. perlata lepida (Pearly Conure)-5; P. hypoxantha sallvadori

(Yellow-sided Conure)-3

Trichoglossus (52) T. haematodus (Green-naped Lorikeets or Rainbow Lory)-49; T. haematodus capistratus (Edward's Lorikeet)-3

Anseriformes Anatidae Anas (3) Anas sp. (Duck)-3

Branta (1) B. sandvicensis (Hawaiian Goose)-1

Cairina (1) C. moschata (Muscovy Duck)-1

Ciconiiformes Ardeidae Nycticorax (1) N. nycticorax (Black-crowned Night Heron)-1

Ciconiidae Ciconia (256) C. ciconia (White Stork)-256

Columbiformes Columbidae Columba (2) Columba livia (Pigeon)-2

Cuculiformes Musophagidae Tauraco (10) T. persa (Guinea Turaco)-4; T. livingstonii (Livingstone's Turaco)-5; T. hartlaubi (Hartlaub's Turaco)-1

Musophaga (7) M. violacea (Violet Turaco)-7

Galliformes Phasianidae Lophura (1) L. nycthemera (Silver Pheasent)-1

Gallus (5) G. domesticus (Chicken and Chicken Silkie Bantom)-5

Coturnix (3) C. japonica (Japanese Quail )-3

Pavo (2) P. cristatus (Common Pea Fowl)-2

Gruiformes Gruidae Grus (14) G. japonensis (Red Crowned Crane)-14

Passeriformes Estrildidae Padda (5) P. oryzivora (Java Sparrow)-5

Fringillidae Coccothraustes (1) C. coccothraustes (Hawfinch)-1

Lagopus (1) L. mutus (Rock Ptarmigan)-1

Hirundinidae Hirundo (1) H. rustica (Barn Swallow)-1

Ploceidae Passer (1) P. montanus (Eurasian Tree Sparrow)-1

Pycnonoyidae Hypsipetes (1) H. amaurotis (Brown-eared Bulbul )-1

Sturnidae Gracula (2) G. religiosa (Southern Grackle or Hill Mynah)-2

Sturnus (1) S. cineraceus (Grey Starling)-1

Piciformes Ramphastidae Ramphastos (11) R. toco (Toco Toucan)-3; R. tucanus (Red-billed Toucan)-3; R. sulfuratus (Keel-billed Toucan)-1; R. vitellinus (Channel-billed Toucan)-3;

R. swainsonii (Chestnut-mendibled Toucan)-1

Sphenisciformes Sphaniscidae Spheniscus (5) S. demersus (Cape Penguin)-5

Strigiformes Strigidae Bubo (5) B. bengalensis (Bengal Eagle Owl)-3; B. virginianus (Horned Owl)-2

Tytonidae Tyto (1) T. alba (Barn Owl)-1

a Common names of avian species are shown in the parentheses.

Table 2. Type of avian fauna screened and distribution according to clinical history and sources of sampling.

Order Family Genus Total samples

(sample no.) (sample no.) Normal Sicke Dead Normal Sicke Dead Normal Sicke Dead Normal Sicke Dead

Psittaciformes (806) Cacatuidae (184) Cacatua 7 0 0 91 0 0 0 0 0 0 0 0 98

Eolophus 3 0 0 4 0 0 1 0 1g 0 0 0 9

Nymphicus 24 10 0 36 0 0 7 0 0 0 0 0 77

Psittacidae (622) Agapornis 7 3 0 1 0 0 0 0 0 0 0 0 11

Amazona 4 1 0 8 0 0 0 0 0 0 0 0 13

Ara 5 4 0 12 0 0 7 20 7f 0 0 0 55

Aratinga 1 0 0 42 0 0 1 0 0 0 0 0 44

Bolborhynchus 0 0 0 1 0 0 0 0 0 0 0 0 1

Brotogeris 0 0 0 2 0 0 0 0 0 0 0 0 2

Cyanoliseus 0 0 0 1 0 0 0 0 0 0 0 0 1

Eclectus 0 0 0 9 0 1 0 0 0 0 0 0 10

Eos 0 0 0 3 0 0 0 0 0 0 0 0 3

Forpus 0 1 0 0 0 0 0 0 0 0 0 0 1

Lorius 0 0 0 11 0 0 0 0 0 0 0 0 11

Melopsittacus 15 25 4 1 0 0 14 0 0 0 0 0 59

Myiopsitta 0 0 0 1 0 0 0 0 0 0 0 0 1

Neophema 0 1 0 1 0 0 0 0 0 0 0 0 2

Pionites 2 1 0 10 0 1 0 0 0 0 0 0 14

Pionus 3 0 0 9 0 0 0 0 0 0 0 0 12

Platycerus 0 0 0 1 0 0 0 0 0 0 0 0 1

Poicephalus 1 0 0 118 2 0 0 0 0 0 0 0 121

Polytelis 0 0 0 3 0 0 0 0 0 0 0 0 3

Pseudeos 0 0 0 7 0 0 0 0 0 0 0 0 7

Psittacula 0 0 0 12 0 0 0 0 0 0 0 0 12

Psittacus 6 1 0 154 1 0 1 0 0 0 0 0 163

Pteroglossus 0 0 0 2 0 0 0 0 0 0 0 0 2

Pyrrhura 2 1 0 18 0 0 0 0 0 0 0 0 21

Trichoglossus 0 0 0 6 0 0 29g 4 13g 0 0 0 52

Anseriformes (5) Anatidae (5) Anas 1 0 0 0 0 0 2 0 0 0 0 0 3

Branta 1 0 0 0 0 0 0 0 0 0 0 0 1

Cairina 1 0 0 0 0 0 0 0 0 0 0 0 1

and wild faunad

Bird sanctuaries

Source and clinical status wise division of the samples

Classification of the screened avian fauna Veterinary hospitalsa Bird parks and zooscPet shopsb

Ciconiiformes (257) Ardeidae (1) Nycticorax 0 0 0 0 0 0 0 0 0 0 1 0 1

Ciconiidae (256) Ciconia 0 0 0 0 0 0 0 0 0 254 0 2 256

Columbiformes (2) Columbidae (2) Columba 0 0 0 0 0 0 2 0 0 0 0 0 2

Cuculiformes (17) Musophagidae (17) Tauraco 0 0 0 0 0 0 10 0 0 0 0 0 10

Musophaga 0 0 0 0 0 0 7 0 0 0 0 0 7

Galliformes (11) Phasianidae (11) Lophura 0 0 0 0 0 0 1 0 0 0 0 0 1

Gallus 3 0 0 0 0 0 2 0 0 0 0 0 5

Coturnix 0 0 0 0 0 0 3 0 0 0 0 0 3

Pavo 0 0 0 0 0 0 2 0 0 0 0 0 2

Gruiformes (14) Gruidae (14) Grus 0 0 0 0 0 0 14 0 0 0 0 0 14

Passeriformes (13) Estrildidae (5) Padda 4 0 0 0 0 0 1 0 0 0 0 0 5

Fringillidae (2) Coccothraustes 0 0 0 0 0 0 0 0 0 0 1 0 1

Lagopus 0 0 0 0 0 0 0 0 0 0 0 1 1

Hirundinidae (1) Hirundo 0 0 0 0 0 0 1 0 0 0 0 0 1

Ploceidae (1) Passer 1 0 0 0 0 0 0 0 0 0 0 0 1

Pycnonoyidae (1) Hypsipetes 1 0 0 0 0 0 0 0 0 0 0 0 1

Sturnidae (3) Gracula 1 0 1 0 0 0 0 0 0 0 0 0 2

Sturnus 0 0 0 0 0 0 0 0 0 0 1 0 1

Piciformes (11) Ramphastidae (11) Ramphastos 0 0 0 0 0 0 11 0 0 0 0 0 11

Sphenisciformes (5) Sphaniscidae (5) Spheniscus 0 0 0 0 0 0 5 0 0 0 0 0 5

Strigiformes (6) Strigidae (5) Bubo 0 0 0 0 0 0 5 0 0 0 0 0 5

Tytonidae (1) Tyto 0 0 0 0 0 0 1 0 0 0 0 0 1

Total 93 48 5 564 3 2 127 24 21 254 3 3 1147

a Include samples from hospitals located in Nara, Tokyo, Gifu, Aichi, Saitama, Okinawa, Kanagawa, Kyoto and Fukuoka prefectures of Japan and represents birds those were

individually or pair wise caged and kept as pets in households. b Mostly birds were imported from South Africa, Singapore, Thailand and USA or in house bred by pet shops (located at Tokyo, Aichi, Osaka and Hyogo prefectures) and sold to

various clients. c Include birds kept in large enclosures as colonies in artificial habitats and are exposed to human contact continuously. These bird parks or zoos are located in Okinawa, Hokkaido

(Kushiro), Hyogo and Shimane prefectures of Japan.d The samples represent birds living in natural or near natural habitats and also includes oriental white storks imported from Russia. Samples were collected from Hokkaido and Gifu

prefectures. e The birds showing the chlamydia specific or unrelated clinical signs and are not clinically normal. f Include samples from outbreak-I in a bird park at Shimane prefecture.g Include samples from outbreak-II in another bird park at Okinawa prefecture.

14

CMGP-2F (5'-GCCTTAAACATCTGGGATCG-3'; nt 384 to 403), CMPG-2R (5'-

GCACAACCACATTCCCATAAAG-3'; nt 634 to 613) was used for the first and second

steps, respectively. Five µl of the first step PCR product was used for the second step PCR.

This test is able to detect 2 to 10 copies of genomic DNA of diverse types of

Chlamydophila sp. in a 50 µl reaction mixture (data not shown). The primers were

synthesized by the Rikaken Co., Nagoya, Japan. In both steps, reaction was performed in

50 µl reaction mixture containing 0.15 µM of each forward and reverse primer, 250 µM of

each dATP, dTTP, dGTP, dCTP, 100 µM of Mg2+

in buffer and 2.5 units of TaKaRa Ex-

Taq (Takara Bio Inc., Otsu, Shiga, Japan) and 2.0 to 5.0 µg of template DNA of each

sample. The thermo cycling conditions used were initial denaturation at 94°C for 3 min,

then 35 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec and

extension at 72°C for 60 sec, then final extension for 5 min at 72°C and soaking at 4°C. In

the second step PCR; the same thermal cycle conditions were used except shorter

extension period of 30 sec. The DNA of C. caviae was used as positive control and PBS

(pH 7.4) and water were used as negative controls.

Cloning of PCR product and sequencing: The PCR products after second step

were purified by gel electrophoresis using low melting agarose gel in Tris-acetate-EDTA

(TAE) buffer, pH 7.4 followed by QIAquick Gel Extraction kit (Qiagen, Hilden,

Germany). The DNA fragment was cloned in pGEM-T vector (Promega, Madison,

Wisconsin) and DH5! strain of E. coli (Tyobo Co., Osaka, Japan) was used for

transformation by heat shock method (171). DNA insert containing clones were selected

on Luria-Bertani (LB) medium plates containing isopropyl-"-D-thiogalactopyranoside

(IPTG) (23.8 µg/ml), ampicillin (50 µg/ml) and 5-Bromo-4-Chloro-"-D-Galactoside (X-

gal) (40 µg/ml) (171). From each sample, 3 to 5 clones with expected size DNA insert

were taken for sequencing. We preferred cloning and sequencing over direct sequencing

15

because DNA of normal intestinal flora (fecal samples) interfered with direct sequencing.

The sequencing was done using the dye-terminator method and performed by a

commercial resource (Dragon Genomics Co., Yokkaichi, Mia, Japan). Both strands were

read. The sequences were assembled and edited using Genetyx-Mac/ATSQ 4.2.3 and

Genetyx-Mac, version 13.0.6 (SDC, Tokyo, Japan).

Analysis of sequences and construction of phylogenetic trees: The chlamydia

species and strains were identified by NCBI-BLAST (http://www.ncbi.nlm.nih.gov) search

of nucleotide and deduced amino acids sequences. For phylogenetic analysis, the ompA

gene sequences of C. psittaci strains and each representative species of genus

Chlamydophila as well as Chlamydia trachomatis were retrieved from the DNA Data Bank

of Japan (DDBJ). Multiple alignments of the trimmed sequences were done using

ClustalX, version 1.83 (198).

Phylogenetic analysis was done with programs in the Phylogeny Inference Package

software (PHYLIP) (version 3.6a3; [http://evolution.genetics.washington.edu

/phylip.html]). The distance matrix between species was computed by DNADIST using

F84 model (46, 102) and clustering of lineages was done by NEIGHBOR using neighbor-

joining method (170). The phylogenetic tree of inferred amino acid sequences was also

constructed by neighbor-joining method with the distance matrix calculated using

PROTDIST by Jones-Taylor-Thornton model (91). The bootstrap values were calculated to

evaluate the branching reliability of trees from a consensus tree constructed by generating

1,000 random data sets using SEQBOOT (45). C. trachomatis strain D/IC-Cal-8 (accession

no. X62920) was used as out-group.

16

RESULTS

The epizootiological and genetic data were analyzed to identify the chlamydial

genotypes prevalent among different avian species kept or living in different habitats and

having variable disease manifestations. Out of 1,147 samples screened, 68 (5.93%) were

found positive by the nested PCR among clinically normal, sick and dead birds of all avian

species (Table 3). By sequencing and analyzing the genetic composition of VD2 region of

ompA gene, genetic diversity and phylogenetic relationship among detected strains of

chlamydiae was examined.

Genetic variation and relative prevalence of chlamydial species and strains:

The genetic analysis of VD2 region of ompA genes from 68 samples showed prevalence of

64 C. psittaci (94.12%), 3 C. abortus (4.41%) and 1 unknown type of Chlamydophila sp.

(1.47%) among all avian species (Table 3).

Table 3. Prevalence of chlamydiae according to the sampling sources and clinical status of the host avian

species.

Source Clinical status wise prevalence rate Overall Chlamydial

positive species

(sample no.)

Normal Sicka Dead

Vet. hospitals 2/93 (2.15%)b 7/48 (14.58%) 2/5 (40%) 11/146 (7.53%) C. psittaci (11)

Pet shops 30/564 (5.32%) 0/3 (0%) 1/2 (50%) 31/569 (5.45%) C. psittaci (31)

Bird parks and 6/127 (4.72%) 0/24 (0%) 12/21 (57.14%) 18/172 (10.46%)c C. psittaci (17)

zoos C. abortus (1)

Bird sanctuaries 8/254 (3.14%) 0/3 (0%) 0/3 (0%) 8/260 (3.08%) C. psittaci (5)

and wild fauna C. abortus (2)

Unknown

Chlamydophila

species (1)

Total 46/1038 (4.43%) 7/78 (8.97%) 15/31 (48.39%) 68/1147 (5.93%)

a Indicate the birds showing the chlamydia specific or unrelated clinical signs.

b The figures are positive samples/total tested samples. The positive percentage is shown in parentheses.

c Includes samples of two outbreaks of avian chlamydiosis.

17

Among sequenced samples, 5 kinds of nucleotide sequences (designated as VD2

sequence types) were detected in VD2 region of C. psittaci ompA gene (Fig. 1 and 2; VD2

sequence types 1, 4, 5, 7 and 8 in Table 4). All detected sequence types of C. psittaci were

grouped into 4 genetic clusters, named from I to IV, based on the genetic distance ! 0.014

(Table 4). The genetic distance was calculated by PHYLIP. The majority of detected

strains belong only to clusters I (57.35%) and II (19.12%) and these strains were detected

mainly among psittacine species and only 2 oriental white storks (Fig. 3). On the basis of

phylogenetic and genetic distance analysis, all the known (from data bank) and detected

strains of C. psittaci were divided into 10 genetic clusters (named I to X) (Table 4 and Fig.

4).

The nucleotide and amino acid sequence based percent identity matrix (Table 5)

showed that all the detected and known strains of C. psittaci can be broadly divide into two

large groups. The first group is represented by the strains of clusters I to III and IX (Table

4). The second group is represented by the strains of clusters IV, V, VI, VII, VIII and X

and also CPX0308 strain (Table 4), those are genetically close to C. abortus (Fig. 4). The

WC and CPX0308 strains appeared to be genetically close to the second group of strains.

WC strain showed 88% amino acid homology with GD strain, whereas, CPX0308 showed

79% nucleotide and amino acid homology with C. caviae-GPIC and C. felis-FP baker

strains, respectively.

Phylogenetic analysis: Nucleotide sequence based neighbor-joining (NJ)

phylogenetic tree showed distinct genetic clustering of C. psittaci strains (Fig. 4). CA0302,

CA0306, CA0307 (Table 4) strains form a cluster along with various strains of C. abortus.

The newly detected strains of clusters III and unknown type of Chlamydophila sp., form

significantly distinct diverging clusters from other strains.

18

Fig. 1. Comparative nucleotide sequence alignment of representative variant strains of C.

psittaci, C. abortus and CPX0308 (detected strains in bold letters) in the VD2 region

(within box) and flanking constant domain region of ompA gene. The representative strains

correspond to each group of strains having 100% nucleotide homology in VD2 region

(designated as VD2 sequence types) as shown in Table 4. The alignment was done by

Genetyx-Mac, version. 13.0.6.

CP0312 1 CTTCGACATTTTCTGCACCTTAGGGGCATCCAATGGATACTTCAAAGCAAGTTCGGCTGCATTCAACTTGGTTGGGTTAATAGGGTTTTCAGCTGCAAGCTCA 103GV 1 ...........A........................................................................................... 1036BC 1 ....................................................................................................... 103CP0435 1 ..............................................T...............................................A.C.A.... 103CP0436 1 ..............................................T...............................................A.C.A.... 103MN 1 ..............................................T...............................................A.C...... 103CP0309 1 ........................A.....................................................................A...C.... 103M56 1 ......T.................A.............................T..C....................................T.....G.. 103CP0303 1 ...T..T...........A.....C..T..T.....G...........T.....T..G.........C.C.....T..G..T..TC..AA..GAA.TGAT.TC 101CA0302 1 ...T..T...........A.....C..T..T.....G...........T.....T..G.........C.C.....T..G..T..TG..AA..GAT.CTC.AT. 103VS225 1 ...T..T...........A.....T..T........G...........T.....T............C.C.....T..G..T..TG..AA..GAA.CTC.GT. 103R54 1 ...T..T...........A.....C..T..T.....G...........T.....C..G.........C.C..C..A..G..T..TG..AA..GA..CTCT.T. 10384/2334 1 ...T..............A.....C..T..T.....C...........T.....C..G........TC.C..C..T..G..T..TA..AA..GAAAC.C..T. 103GD 1 T..T..T...........A.....C..T..T.....G...........T.....T..G........TC.C.....T..G..T..TG..AA..GA---.....C 100WC 1 ...T..T...........A.....T..T..T.....C........G.G......C.....T.....TC.T.....C..GT.C..AA..G.T.GA---..TAGC 100CPX0308 1 T.....TG....T.....T.....T..TA.T.....C..T..T...T.T.A...T..A..T..T.GTC.T.....C.....T..AG..G.T.GA.G.TC..T. 103

CP0312 103 ATCTCTACCGATCTTCCAACGCAACTTCCTAACGTAGGCATTACCCAAGGTGTTGTGGAATTTTATACAGACACATCATTTTCTTGGAGCGTAGGTGCACGTGGAG 209GV 103 .......................................................................................................... 2096BC 103 ...................T...................................................................................... 209CP0435 103 .C.................T...................................................................................... 209CP0436 103 .C...........C.....T...................................................................................... 209MN 103 .C.........G.......T...................................................................................... 209CP0309 103 .C........................................................................................................ 209M56 103 G.TAG..............A.................C............................................................C....... 209CP0303 101 .AT---.ATC.A......------------.......C...C........C.....T..G.....C.........A....C...........G............. 194CA0302 103 GCAG..GATC.G.....C------------..T........C..T.....AA.C..T..............T...A....C........T...........C.... 197V225 103 GCAG..GATC.A......------------...........C..T......A....T..G.....C.....T...A....C.......................T. 197R54 103 TCA---GAGC.A......------------...........C..T...........T..G.....C.....T...A...................C.....C.... 19484/2334 103 .CAAA.GA.CGA.....C------------...........C..T.....C.....T..G.....C.....T...A....C....................C.... 197GD 100 T.AA.A.AT..C---------........C.......C...C..T.....C.....T..G.....C.....T...A.G..C......................... 197WC 100 GAAAG..AT.C....AATGAC........A.......CT..C..A.....AA.C..T..G.....C.....T..CA....C...........G........C.... 206CPX0308 103 .ATG...AT..A.....T------------......TT...G..A......A....A..GC....C..T......A....C...........T............. 197

Variable domain 2 Constant domain

Constant domain Variable domain 2

Constant domain Variable domain 2 Constant domain

CP0312 1 FDIFCTLGASNGYFKASSAAFNLVGLIGFSAASSISTDLPTQLPNVGITQGVVEFYTDTSFSWSVGARG 69 GV 1 ...Y................................................................. 69 6BC 1 ........................................M............................ 69 CP0435 1 ...............S...............TN.T.....M............................ 69 CP0436 1 ...............S...............TN.T.....M............................ 69 MN 1 ...............S...............T..T..E..M............................ 69 CP0309 1 ...............................TT.T.................................. 69 M56 1 ...............................S.AV.....K.....A...................... 69 CP0303 1 ............................LKGTDFN-NQ..----..A............T......... 64 CA0302 1 ............................VKGS.IAADQ..----.......I.......T......... 65 VS225 1 ............................VKGT.VAADQ..----.......I.......T......... 65 R54 1 ............................VKG..LS-EQ..----...............T......... 64 84/2334 1 ............................IKGNTLTNDR..----...............T......... 65 GD 1 ............................VKGS.LTND----.....A............T......... 65 WC 1 ...............G..........F.IAGN.E-.NA.ND.....A....I.......T......... 68 CPX0308 1 ..V......T.....SN....S......VAGG.LNANE..----..FM...I..L....T......... 65

Fig. 2. Amino acid homology among representative variant strains of C. psittaci, C. abortus and CPX0308 (detected strains in bold letters)

in the VD2 region of ompA gene. The portion within the box represents the genetically variable domain 2 (VD2) flanked by the constant reg-

-ions of ompA gene. Each representative strain corresponds to a group of strains having 100% nucleotide homology in VD2 region as shown

in Table 4. The alignment was done by Genetyx-Mac, version 13.0.6.

Table 4. Clusters of detected and known strains of C. psittaci based on nucleotide sequence similarity in the VD2 region of the ompA gene.

Genetic clusters VD2 Chlamydial

(! 0.014 genetic sequence species

distance) typesa Name No. Name (accession no.) No.

I 1 CP0312b, CP0315, CP0316, CP0317, 39 MN Zhang (AF269281), 90/1051 (AY762608) 2 C. psittaci

CP0318, CP0320, CP0321, CP0322,

CP0323, CP0324, CP0325, CP0326,

CP0327, CP0328, CP0329, CP0330,

CP0331, CP0369, CP0370, CP0371,

CP0372, CP0373, CP0374, CP0375,

CP0376, CP0432, CP0434, CP0448,

CP0449, CP0450, CP0451, CP0452,

CP0453, CP0454, CP0455, CP0456,

CP0457, CP0458, CP0462

2 Not detectedc 6BCb (X56980) 1 C. psittaci

3 Not detectedc GVb (L25437) 1 C. psittaci

II 4 CP0435b, CP0437, CP0438, CP0439, 11 CP3 (AF269265), 41A12 (AY762609) 2 C. psittaci

CP0440, CP0441, CP0443, CP0444,

CP0445, CP0446, CP0447

5d CP0436b, CP0442 2 Not reportede 0 C. psittaci

6 Not detectedc MNb (AF269262), MNOs (AF269264), 8 C. psittaci

MNRh (AF269263), A22/M (M36703),

WS/RT/E30 (AY762613), 3759/2 (AY762611),

N352 (L04980), 98AV2129 (AY327465)

III 7 CP0309b, CP0311, CP0314, CP0319 7 Not reportede 0 C. psittaci

CP0459, CP0460, CP0461

IV 8 CP0303b, CP0304, CP0305, CP0310, 5 NJ1 (AF269266), TT3 (AF269267), 3 C. psittaci

CP0313 7344/2 (AY762610)

V 9 Not detectedc GDb (AF269261), CT1 (AF269260), 3 C. psittaci

Avian type C (L25436)

VI 10 Not detectedc VS225b (AF269259), 7778B15 (AY762612) 2 C. psittaci

VII 11 Not detectedc 84/2334b (AJ310735) 1 C. psittaci

VIII 12 Not detectedc R54b (AJ243525) 1 C. psittaci

IX 13 Not detectedc M56b (AF269268) 1 C. psittaci

X 14 Not detectedc WCb (AF269269) 1 C. psittaci

15 CA0302b, CA0306K, CA0307K 3 B577 (M73036), EBA (AF269256), 9 C. abortus

OCHL196 (AJ004873), BA1 (L39020),

pm326 (AJ004875), LW508 (M73040),

S26/3 (X51859), pm225 (AJ005617),

wt parakeetf

16 CPX0308b 1 Not reportede 0 Unknown

Chlamydophila

species

a Detected or known strains having 100% nucleotide homology in VD2 region of ompA gene are designated as VD2 sequence type.b The representative strains used for analysis in Fig. 1, Fig. 2 and Table 5. c Strains not detected in our study.d Having 1 nucleotide difference in the VD2 as compared to VD2 type 4 sequences but no change in amino acid residue.e Strains not reported in DNA data banks.f The ompA gene of the strain isolated from a diseased parakeet was found 100% homologous to B577 strain (98).

Detected strains Known strains

Table 5. Percentage identity matrix (PIM) of detected strains (representative strains only) and known strains of C. psittaci and C. abortus in VD2 region of ompA gene.

VD2 Genetic

types clusters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

1: C. psittaci-CP0312 1 100 100 100 97 97 97 98 94 79 80 79 77 78 77 73 72 70 72 69 68 60

2: C. psittaci-GV 3 99 100 99 97 96 97 98 93 79 79 79 77 78 77 72 72 70 72 68 68 60

3: C. psittaci-6BC 2 99 97 100 98 97 98 98 94 79 80 79 77 78 77 73 72 70 72 69 68 60

4: C. psittaci-CP0435 4 93 91 94 100 100 99 98 92 80 80 79 78 79 77 72 72 70 73 69 67 61

5: C. psittaci-CP0436 5 93 91 94 100 100 99 97 91 80 80 79 78 79 77 72 72 70 73 69 67 61

6: C. psittaci-MN 6 93 91 94 97 97 100 97 92 79 80 79 78 79 77 72 71 69 72 68 66 60

7: C. psittaci-CP0309 7 III 96 94 94 96 96 94 100 93 88 81 79 78 79 78 73 72 70 73 70 69 61

8: C. psittaci-M56 13 IX 93 91 93 90 90 90 91 100 88 80 77 77 75 78 74 72 72 73 71 70 60

9: C. psittaci-CP0303 8 IV 86 83 86 86 86 84 80 79 100 91 91 92 90 88 81 76 78 80 75 71 64

10: C. psittaci-VS225 10 VI 85 83 85 85 85 85 86 83 92 100 92 91 89 97 81 77 75 78 74 69 65

11: C. psittaci-R54 12 VIII 91 89 91 88 88 88 89 86 93 92 100 91 91 92 79 74 74 79 72 69 66

12: C. psittaci-GD 9 V 85 83 85 83 83 83 85 86 95 91 94 100 92 92 81 77 75 76 73 72 64

13: C. psittaci-84/2334 11 VII 83 82 83 82 82 82 83 82 92 88 91 92 100 88 83 73 73 75 71 70 63

14: C. abortus-CA0302 15 - 86 83 86 83 83 83 85 83 90 97 92 92 88 100 79 73 73 76 71 70 63

15: C. psittaci-WC 14 X 79 76 79 79 79 79 79 79 86 86 86 88 86 86 100 76 70 74 73 69 63

16: Unknown type-CPX0308 16 - 71 69 71 72 72 72 71 69 77 78 78 77 74 78 75 100 79 73 71 72 62

17: C. caviae-GPICa - - 73 70 73 73 73 72 75 75 78 79 79 79 74 77 75 74 100 77 70 72 65

18: C. felis-FP Bakera - - 77 73 77 77 77 75 78 77 84 84 80 79 77 82 77 79 78 100 70 70 64

19: C. pneumoniae-CSFa - - 69 65 69 69 69 69 69 70 70 71 72 76 76 73 73 69 66 66 100 70 67

20: C. pecorum-1710Sa - - 66 67 66 68 68 68 68 68 74 72 70 69 69 69 68 74 74 76 69 100 65

21: C. trachomatis-Da - - 57 54 57 57 57 57 57 57 59 59 62 59 61 59 60 64 62 59 64 64 100

a The accession no. of sequences are: C. caviae-GPIC (AF269282), C. felis-FP Baker (AF269257), C. pneumoniae-CSF (AF131889), C. pecorum-1710S (AF269279) and

C. trachomatis-D (X62920). For C. psittaci strains accession no. are shown in Table 4.

The upper right triangular half of the matrix is nucleotide based and lower left triangular half is amino acid based. The bold figures separating two triangular halves indicate the

100% identity based on nucleotide and amino acid sequences. The strains having !90% identity are boxed together in the solid line. The numbers (1 to 21) in the top row

correspond to the respective species/strains listed in the first column. The matrix was constructed by ClustalX (version 1.83). The comparative PIM of C. psittaci strains with

other Chlamydophila species and C. trachomatis infecting mammals and human (non-avian hosts) is also shown.

I

II

23

Fig. 3. Diagram showing the comparative prevalence of chlamydial strains detected in this study.

The types of avian species (psittacine species are members of Cacatuidae and Psittacidae families)

tested chlamydia positive are shown in boxed text near each cluster of strains. Two largest clusters

of C. psittaci strains are cluster I (57.35%) and cluster II (19.12%) respectively.

Epizootiological analysis: The cases of avian chlamydiosis were mainly due to C.

psittaci strains and detected among 58 samples from Psittaciformes birds, 5 from oriental

white storks and 1 from Java sparrow. The C. abortus was detected in 1 budgerigar and 2

oriental white storks samples. One unknown species, CPX0308 was also found in an

oriental white stork imported from Russia to a bird sanctuary in Japan (Table 6). The

strains of cluster I were mainly detected in chlamydiosis cases including 2 outbreaks (Fig.

10 psittacine species,

1 oriental white stork

5 psittacine

species,

1 oriental

white stork

2 oriental

white storks,

2 budgerigars,

1 Java

sparrow

2 oriental white storks,

1 budgerigar

1 oriental white stork

15 psittacine

species,

1 oriental

white stork

24

5). However, other genotypes were also detected occasionally including an outbreak with

strains of cluster III (mixed infection with cluster I strains). The month wise incidence rate

of chlamydiosis due to various genotypes during 2003 and 2004 is shown in Fig. 5. The

chlamydiosis cases were detected throughout year but slight rise was observed in

chlamydiosis incidences in the beginning of winter and spring seasons including 2

outbreaks.

The prevalence rate according to clinical status, sources of sampling, avian species,

sex, age and geographical locations is as follow:

(i) Clinical status wise prevalence. Among 4.43% (46/1038) clinically normal birds

all 6 types of C. psittaci genotypes (genetic clusters) were detected in the feces including

one case (CP0432) in which only blood sample gave positive PCR result. The strains of

clusters I and II were detected from 8.97% (7 out of 78) sick birds showing chlamydia

specific and non-specific symptoms. 48.39% (15 out of 31) dead birds, those showed

typical clinical symptoms of chlamydiosis before death and histopathological lesions, were

found to carry chlamydial strains of clusters I, II and III (Tables 3 and 6).

(ii) Avian habitat wise prevalence. According to the habitats of avian hosts

(represented by the sources of sampling) that also epitomize managemental conditions and

avian-human interactions, the highest 10.46% cases were detected from bird parks/zoos

(including 2 outbreaks by strains of clusters I and III), followed by 7.53% from

individually/pair wise caged birds, mostly kept as pets in households. However, 5.45% and

3.08% cases were detected respectively from pet shops and bird sanctuaries (Table 3). The

cluster II strains were detected mostly from one pet shop. The strains of cluster I and II

were detected most often among samples from veterinary hospitals and pet shops. The

chlamydial infection in the bird parks and zoos were mostly due to strains of clusters I, III,

and IV of C. psittaci and C. abortus strains, whereas, among samples from bird sanctuaries

25

Fig. 4. Neighbor-joining (NJ) phylogenetic tree, based on nucleotide sequences of VD2

region of ompA gene of different strains of C. psittaci, C. abortus and other

Chlamydophila species. The strains detected in this study are shown in bold letters and

vertical lines mark genetic clusters of C. psittaci strains from I to X. The genetic

distance is indicated in 0.05 unit bar. Bootstrap values are shown at respective nodes.

The C. trachomatis is used as out-group.

WC (AF269269)

B577 (M73036) pmSH1 (AJ005618) pm234 (AJ004874) pmd623 (AJ005615) pm112 (AJ005613)

EBA (AF269256) BA1 (L39020) S26/3 (X51859) LW508 (M73040) pm225 (AJ005617) pm326 (AJ004875) OCHL196 (AJ004873) CA0302 CA0306 CA0307

VS225 (AF269259) 7778B15 (AY762612)

GD (AF269261) CT1 (AF269260) Avian type C (L25436)

84/2334 (AJ310735) NJ1 (AF269266) TT3 (AF269267) 7344/2 (AY762610) CP0303 CP0304 CP0305 CP0310 CP0313

R54 (AJ243525)

C. felis-FP Baker (AF269257)

M56 (AF269268) CP0309 CP0311 CP0314 CP0319

CP3 (AF269265) CP0436 CP0442

41A12 (AY762609) CP0435 CP0437 CP0438 CP0439 CP0440 CP0441 CP0443 CP0444 CP0445 CP0446 CP0447 MN (AF269262) MNOs (AF269264) MNRh (AF269263)

GV (L25437)

A22/M (M36703) N352 (L04980) 3759/2 (AY762611) WS/RT/E30 (AY762613) 98AV2129 (AY327465)

6BC (X56980)

CP0359 CP0360 CP0361

CP0312 CP0315 CP0316 CP0317 CP0318 CP0320 CP0321 CP0322 CP0323 CP0324 CP0325 CP0326 CP0327 CP0328 CP0329 CP0330 CP0331 CP0369 CP0370 CP0371 CP0372 CP0373 CP0374 CP0375 CP0376 CP0432 CP0434 CP0448 CP0449 CP0450

CP0452 CP0453 CP0454 CP0455

90/1051 (AY762608) MN Zhang (AF269281)

CP0456 CP0457 CP0458 CP0462

C. caviae-GPIC (AF269282) CPX0308

C. pneumoniae-CSF (AF131889)C. pecorum-1710S (AF269279)

C. trachomatis-D/IC-Cal-8 (X62920)

C. a

bort

us

I

II

III

IV

100%

100%

99.9%

100%

95.5%

90.5%

79.4%

0.05 units

CP0451

V

VI

VII

VIII IX

X

Fig. 5. Month wise incidences of avian chlamydiosis cases detected by PCR during 2003 and

2004 among all avian species. The numerical figures shown inside and above the bars are actu-

-al numbers of chlamydiae positive and negative cases. The numbers shown in columns below

each bar indicatethe distribution of samples and detected strain type in each month. Outbreak-I

and outbreak-II took place at two different bird parks and boxes mark the types of C. psittaci

strains involved.

I. 0 0 1 0 0 1 3 1 6 7 8 8 1 1 0 0 0 0 1 0 0 0 1 0

II. 0 0 0 0 0 0 0 0 0 0 0 0 0 2 11 0 0 0 0 0 0 0 0 0

III. 0 0 3 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 3 0

IV. 0 3 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Genetic clusters

(2004)(2003)

outbreak-I

outbreak-II

Sam

ple

num

ber

0

62

40

5951

27

60

36

6574

37

140

4136

151

32

18

47

9 1217

54

0

0

1

1

11

0

0

0

1

00

0

47

4

11

8

3

9

7

6

3

6

6

0

0

20

40

60

80

100

120

140

160

180

Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

Sample numbers

Positive

Negative

Number of samples

C. psittaci

C. abortus

Unknown

Chlamydophila sp.

28

strains of clusters I, III, IV and unknown type of the Chlamydophila sp. were detected

(Table 6).

(iii) Avian species wise prevalence. The incidences of chlamydiosis were

predominantly detected among Psittaciformes birds (detected among 28 psittacine avian

species). It includes chlamydia positive rates of 7.61% (14 out of 184) in family

Cacatuidae and 7.23% (45 out of 622) in family Psittacidae. However, some psittacine

bird species such as Trichoglossus haematodus 12 out of 52 (23.08%), Nymphicus

hollandicus 11 out of 77 (14.28%), Aratinga sp. 5 out of 44 (11.36%), Ara sp. 5 out of 55

(9.09%) and Mesopsittacus undulates 5 out of 59 (8.47%) showed high incidences (Tables

1, 2 and 6). In the Cacatuidae family mainly the cluster I strains were detected; however,

in Psittacidae, strains of clusters I, II, III, IV and C. abortus were detected. Whereas,

among non-psittacine species, 3.12% (8 out of 256) samples from oriental white storks

(Ciconia boyciana) belonging to Ciconiidae family of order Ciconiiformes, were found

having strains of clusters I, II, III, IV, C. abortus and unknown type of Chlamydophila sp.

A single case of chlamydiosis due to a strain of cluster IV was also detected in a Java

sparrow (Padda oryzivora) belonging to Estrildidae family of Passeriformes order (Table

6).

(iv) Avian sex and age wise prevalence. The incidences of chlamydiosis were found

higher among birds around the age group of 3 months due to different genotypes,

irrespective of their sexes (Table 6).

(v) Worldwide locations wise prevalence. All type of genotypes were detected

among the avian species imported to Japan from Singapore, Indonesia, USA, South Africa

and Russia as well as from different places in Japan, indicating ubiquitous presence of

various chlamydia genotypes infecting different avian fauna (Table 6).

Table 6. Types of chlamydial species/strains detected in relation to the zoological position, sampling source, clinical status, sex, age and import source of avian host species

Gen-

Order, family and scientific name (common name) Species wise Strains etic Accession no.

of the host avian species positive/tested No. Type Clinical history Source Age:sex of the hosta Source of Chlamydial designated clust-

(no. of positive/tested samples) samples (M: male, F: female) importb species ters

Order-Psittaciformes (59/806)

Family-Cacatuidae (14/184)

Cacatua moluccensis (Salmon-crested Cockatoo) 1/18 1 Clotted blood Normal Pet shop 6 months Singapore C. psittaci CP0432 I AB239867

C. sulphurea (Yellow-crested Cockatoo) 1/26 1 Cloacal swab Normal Pet shop Indonesia C. psittaci CP0457 I AB239878

Eolophus roseicapillus (Roseate Cockatoo or Galah) 1/9 1 Pooled visc. orgn.c Dead (outbreak-I)d Bird park/zoo C. psittaci CP0312 I AB239842

Nymphicus hollandicus (Cockatiel) 11/77 2 Feces Normal Vet. Hospital 3 months C. psittaci CP0315 I AB239843

C. psittaci CP0316 I AB239844

4 Feces Sicke Vet. Hospital 6 months C. psittaci CP0322 I AB239848

2 years 10 months C. psittaci CP0460 III AB239897

C. psittaci CP0448, CP0449 I AB239869, AB239870

5 Cloacal swabs Normal Pet shop Domestically C. psittaci CP0451, CP0452 I AB239872, AB239873

bred (Japan) CP0453, CP0454 AB239874, AB239875

CP0455 AB239876

Family-Psittacidae (45/622)

Amazona aestiva (Blue-fronted Amazon) 1/6 1 Feces Sicke Vet. hospital 1 year C. psittaci CP0462 I AB239880

Ara ararauna (Blue-and-yellow Macaw) 1/11 1 Cloacal swab Normal Pet shop C. psittaci CP0444 II AB239888

A. auricollis (Yellow-collard Macaw)* 2/23 2 Cloacal swabs Normal Pet shop 2 years Singapore C. psittaci CP0445, CP0446 II AB239889, AB239890

A. severa (Chestnut-fronted Macaw)* 2/7 2 Pooled visc.orgn.c Dead (outbreak-I)d Bird park/zoo C. psittaci CP0311, CP0314 III AB239893, AB239894

Aratinga aurea (Peach-fronted Conure) 1/3 1 Cloacal swab Normal Pet shop 1 year:F South Africa C. psittaci CP0328 I AB239855

A. pertinax (St. Thomas/Brown-throated Conure)* 1/1 1 Cloacal swab Normal Pet shop 1 year:M South Africa C. psittaci CP0329 I AB239856

A. acuticaudata (Blue-crowned Conure) 1/7 1 Cloacal swab Normal Pet shop South Africa C. psittaci CP0330 I AB239857

A. wagleri (Scarlet or Red-fronted Conure)* 1/2 1 Cloacal swab Normal Pet shop M South Africa C. psittaci CP0439 II AB239884

A. jandaya (Jandaya Conure)* 1/6 1 Cloacal swab Normal Pet shop 3 months South Africa C. psittaci CP0442 II AB239908

Lorius lory (Black-capped Lory)* 1/5 1 Cloacal swab Normal Pet shop 1 year Singapore C. psittaci CP0441 II AB239886

Melopsittacus undulatus (Budgerigar) 5/59 1 Feces Normal Bird park/zoo C. abortus CA0302 - AB239904

2 Feces Normal Bird park/zoo C. psittaci CP0303, CP0304 IV AB239899, AB239900

1 Spleen Dead Vet. hospital 4 months C. psittaci CP0447 II AB239891

1 Liver Dead Vet. hospital C. psittaci CP0438 II AB239883

Neophema bourkii (Bourke's Parrot) 1/2 1 Feces Sicke Vet. hospital 6 months:M C. psittaci CP0458 I AB239879

Poicephalus rueppellii (Rüppell's Parrot)* 2/7 2 Cloacal swabs Normal Pet shop M South Africa C. psittaci CP0326 I AB239853

South Africa C. psittaci CP0450 I AB239871

P. senegalus (Senegal Parrot) 1/42 1 Cloacal swab Normal Pet shop 3 months South Africa C. psittaci CP0319 III AB239895

P. cryptoxanthus (Brown-headed Parrot)* 2/26 1 Cloacal swab Normal Pet shop 3 months:M South Africa C. psittaci CP0323 I AB239850

1 Cloacal swab Normal Pet shop 3 months:M South Africa C. psittaci CP0436 II AB239907

P. meyeri (Meyer's Parrot/Broun Parrot) 1/19 1 Cloacal swab Normal Pet shop 3 months:M South Africa C. psittaci CP0435 II AB239881

Details about the PCR positive and sequenced samples

P. gulielmi (Red-crowned/Jardine's Parrot) 1/23 1 Cloacal swab Normal Pet shop South Africa C. psittaci CP0443 II AB239887

Pionites leucogaster (White-bellied Caique) 1/8 1 Pooled visc.orgn.c Dead f Pet shop 3 months:M USA C. psittaci CP0461 III AB239898

P. melanocephala (Black-headed Caique) 1/6 1 Cloacal swab Normal Pet shop C. psittaci CP0456 I AB239877

Psittacula derbiana (Derbyan Parakeet) 1/3 1 Cloacal swab Normal Pet shop Domestically C. psittaci CP0327 I AB239854

bred (Japan)

Psittacus erithacus (African Grey Parrot) 3/154 3 Cloacal swabs Normal Pet shop 3 months:M South Africa C. psittaci CP0317, CP0318 I AB239845, AB239846

3 months South Africa C. psittaci CP0434 I AB239868

Pyrrhura hypoxantha sallvadori (Yellow-sided Conure)* 1/3 1 Cloacal swab Normal Pet shop 5 months C. psittaci CP0440 II AB239885

P. perlata lepida (Pearly Conure)* 1/5 1 Feces Sickg Vet. hospital 1 year 9 months:M C. psittaci CP0459 III AB239896

Trichoglossus haematodus (Green-napped Lorikeet) 1+11h/49 8 Pooled visc. Orgn.c Dead (outbreak-II)h Bird park/zoo C. psittaci CP0320, CP0369 I AB239847, AB239859

CP0370, CP0371 AB239860, AB239861

CP0373, CP0374 AB239863, AB239864

CP0375, CP0376 AB239865, AB239866

1 Feces Dead (outbreak-II)h Bird park/zoo C. psittaci CP0324 I AB239851

2 Feces Normal (outbreak-II)h Bird park/zoo C. psittaci CP0321, CP0372 I AB239849, AB239862

1 Cloacal swab Normal Pet shop M C. psittaci CP0325 I AB239852

Order-Ciconiiformes (8/257)

Family-Ciconiidae (8/256)

Ciconia boyciana (Oriental White Stork)* 8/256 2 Feces Normal Bird sanctuary Russia C. abortus CA0306, CA0307 - AB239905, AB239906

1 Feces Normal Bird sanctuary Russia Unknown CPX0308 - AB239931

Chlamydo-

phila sp.

3 Feces Normal Bird sanctuary Russia C. psittaci CP0309 III AB239892

Russia C. psittaci CP0331 I AB239858

Russia C. psittaci CP0437 II AB239882

2 Cloacal swabs Normal Bird sanctuary Russia C. psittaci CP0310, CP0313 IV AB239902, AB239903

Order-Passeriformes (1/13)

Family-Estrildidae(1/5)

Padda oryzivora (Java Sparrow) 1/5 1 Feces Normal Bird park/zoo C. psittaci CP0305 IV AB239901

a Only the available data about age and sex of avian species are shown.b Only the confirmed records about the source of importation and breeding place of host species are shown.c Pooled visceral organs includes pieces lung, liver, spleen and heart.d Samples are from outbreak-I.e Include those birds showing chronic weight loss, yellowish-green diarrhea, anorexia, cachexia and rise in body temperature.f Died with acute symptoms and also found positive for avian polyoma virus by PCR.g Had crop inflammation due to yeast infection along with clinical symptoms of chlamydiosis. h Samples from outbreak-II.

* The avian species detected chlamydia positive first time when compared to referred latest updated list (94).

31

DISCUSSION

C. psittaci mainly infects avian species and also has potential zoonotic importance

(8, 181, 188). In this chapter, we investigated the strain level prevalence of various

chlamydiae among diverse avian fauna and examined various situations those contribute to

the maintenance, precipitation and/or horizontal transmission of infection. Further genetic

and phylogenetic relationships among prevalent species/strains were also examined.

We analyzed partial ompA gene (VD2 region) for identification of chlamydial

species and strains. This locus has been reported to be associated with the phylogenetic

divergence of Chlamydophila spp. and Chlamydia spp. and has been used in other

phylogenetic studies. Although the phylogenetic analyses of conserved 16S rRNA, ompA

and rnpB genes of chlamydiae group are often used to classify and differentiate C. psittaci

strains from other species of family Chlamydiaceae (60, 79, 149, 191, 193, 205), the

analysis of ompA gene is advantageous in epizootiological strain typing due to presence of

strain/serovar/genotype specific motifs in 4 genetically variable domains (12, 86, 148,

225). Sequence analysis of highly conserved 16S rRNA gene may not detect minor strain

variations. Direct sequencing of 16S rRNA gene was also not suited in our study due to the

interference by normal intestinal microflora DNA as we tested many samples of fecal/fecal

swab or intestinal loops origin. By our approach, small DNA fragment of ompA gene can

be conveniently amplified from clinical samples by nested PCR and sequenced even in

lower EB concentration in samples (2 to 10 EBs). This approach of direct strain typing

from clinical samples particularly in large-scale epizootiological/epidemiological studies is

less cumbersome as serotyping and isolation is time consuming.

To survey the host range of C. psittaci, total 113 avian species from 11 avian orders

examined. The chlamydiae were detected among 28 species of psittacine birds and 2

species of non-psittacine birds (oriental white stork and Java sparrow). Out of total 28

32

psittacine bird species 10 were detected first time to harbor chlamydiae in this study along

with 1 oriental white stork species (Table 6). Till date the chlamydiae have been detected

among 460 avian species from 30 avian orders that include 153 Psittaciformes species

(94). Therefore, the chlamydiae have been detected among 163 (47.66%) out of total 342

Psittaciformes species prevalent worldwide including results of present study. Some

species popularly kept as pets such as Trichoglossus haematodus (Green-napped Lorikeet),

Nymphicus hollandicus (Cockatiel), Aratinga sp. (Parakeet or Conure), Ara sp. (Macaw)

and Mesopsittacus undulates (Budgerigar) showed exceptionally high incidences of

chlamydiosis. From the results of this study and earlier reports those documented high

chlamydiosis incidences among Psittaciformes species (18, 69) it appears that

Psittaciformes are predominant reservoirs of chlamydiae among all avian species.

Therefore, Psittaciformes avian species may be responsible for maintenance or

transmission of infection in the population.

Although there are many reports regarding the pigeons as natural carriers of C.

psittaci and source of infection to human being (6, 16, 20, 45), in this study only few

samples from pigeons were tested, those were found negative. As the main thrust of this

study was analyzing samples from captive avian fauna, either imported or indigenously

bred and kept under near natural or caged conditions and are often exposed to human

beings and animal fauna.

C. psittaci strains are divided into 8 serotypes named A to H (4, 56, 206, 207) and 7

genotypes from A to F and E/B (60, 174, 207), based on monoclonal antibody typing and

AluI RFLP pattern of ompA gene respectively. In the present study, clustering pattern of C.

psittaci strains in relation to grouping on the basis of serological and RFLP studies was

also analyzed phylogenetically using nucleotide sequences and deduced amino acid

sequences as described in materials and methods. Genetic cluster I includes C. psittaci

33

strains with ompA gene AluI RFLP pattern and serotype type A; clusters II includes RFLP

types B, E and E/B and serotype types B and E; cluster IV includes RFLP and serotype

type D; whereas, strains of cluster III and CPX0308 are unknown types.

The genetic and phylogenetic analysis of chlamydial strains detected in the

screened avian fauna revealed that VD2 sequence type 1 and 4 form clusters I and II along

with genetically related strains classified as serovars/genotypes-A and B by others (4, 174).

Clusters I and II were found predominantly prevalent especially among Psittaciformes

birds. High prevalence of serotypes/genotypes A and B have also being reported in recent

studies (60, 191, 207).

Cluster III strains were also detected from 6 psittacine birds and 1 oriental white

stork. Cluster III strains have 3 amino acid substitutions in VD2 region as compared to

VD2 sequence types 1 and 4 clusters of strains. Cluster III strains appeared to be recently

evolved from cluster II strains.

Strains of cluster IV which are genetically the same as serovar/genotype-D strains

of C. psittaci (4, 60, 174) were detected from 2 oriental white storks, 2 budgerigars and 1

Java sparrow. These strains were usually detected from non-psittacine avian species and

are highly pathogenic to domesticated poultry (4, 7, 8, 207). Our results indicate that these

strains may have broad host range and may dormantly exist among other free-living avian

fauna those can be potential source of infection to domesticated/commercial poultry.

CA0302, CA0306 and CA0307 strains detected from 3 samples, genetically

resembled to a cluster of C. abortus strains. Similarly a “wt parakeet” strain, isolated from

a diseased parakeet by Kaltenboeck et al. (96), was found to have ompA gene sequence

100% homologous to that of B577 strain of C. abortus (98). The genetic data based on

RFLP pattern and 16S rRNA and 16S-23S rRNA inter genic spacer region sequencing

studies have also indicated that the genetic relatedness among C. psittaci and C. abortus is

34

quite high and there are some isolates having the characteristic of both groups (43, 51, 149,

193, 205). Recent studies have also pointed toward this fact (191). The C. abortus seems to

be latest diverging group still having genetic roots in the avian hosts.

From the epizootiological and genetic data of this study and other reports, it can be

inferred that all chlamydial strains responsible for avian chlamydiosis can be broadly

divided into two groups: major C. psittaci strains and minor C. psittaci strains. The major

group includes those strains which are highly prevalent among avian species, evolutionally

conserved and are genetically adapted to class aves. The examples are strains of clusters I,

II, III and IV (Tables 4 and 5) and serovar/genotypes-A, B, C, D, E and E/B (4, 56, 60,

206, 207). Whereas, the minor group include very rarely detected strains among avian and

mammalian species and genetically intermediate between other mammalian

Chlamydophila species and C. psittaci such as strains of CA0302, CA0306 and CA0307

strains and unknown type of Chlamydophila sp. (CPX0308 strain) (Tables 4 and 5) and

serovar-F, G, H and 84/2334, VS225 strains (4, 56, 206, 207) and R54 strain (79), V448,

V351 strains (191) and Daruma strain (51).

Finally, it can be concluded that incidences of avian chlamydiosis are more among

psittacine birds especially under captive conditions. Though disease incidences are high in

some avian species but the C. psittaci is maintained and propagated in the population by a

large variety of inapparently infected avian fauna. Many strains of C. psittaci are

genetically well adapted to avian fauna and associated with disease or simply co-exist in

dormant stage but only some of the strains are predominantly prevalent among birds.

35

SUMMARY

For studying the genetic diversity and occurrence of Chlamydophila psittaci, a total

of 1,147 samples from 11 avian orders including 53 genera and 113 species of feral and

captive birds were examined using ompA gene based nested PCR. Three types of

chlamydiae: C. psittaci (94.12%), C. abortus (4.41%) and unknown Chlamydophila sp.

(1.47%) were identified among 68 (5.93%) positive samples (Psittaciformes-59,

Ciconiiformes-8 and Passeriformes-1). On the basis of nucleotide sequence variations in

the VD2 region of ompA gene, all 64 detected C. psittaci strains were grouped into 4

genetic clusters. Clusters I, II, III and IV were detected from 57.35%, 19.12%, 10.29% and

7.35% samples respectively. A single strain of unknown Chlamydophila sp. was found to

be phylogenetically intermediate between Chlamydophila species of avian and mammalian

origin. Among Psittaciformes, 28 out of 81 tested species including 10 species previously

unreported were found chlamydiae positive. Chlamydiosis was detected among 8.97% sick

and 48.39% dead birds as well 4.43% clinically normal birds. Therefore, it was concluded

that, though various genetically diverse chlamydiae may have caused avian chlamydiosis,

only a few C. psittaci strains were highly prevalent and frequently associated with

clinical/subclinical infections.

36

Analysis of genetic diversity and molecular phylogeny of the Chlamydophila

psittaci strains prevalent among avian fauna and associated with human

psittacosis.

INTRODUCTION

Chlamydophila psittaci is an obligate intracellular pathogen, mainly causing

clinical or subclinical infections among avian species and is also potentially pathogenic to

other mammals and human beings (8, 181). The disease of avian species is called “avian

chlamydiosis” and human infection is known as “psittacosis” (181). The clinical avian

chlamydiosis is either acute or subacute and characterized by respiratory distress, diarrhea,

ocular and nasal discharge, nervous signs or systemic infection with varying severity and

mortality up to 30% (8). The infectious elementary bodies (EBs) of C. psittaci can survive

for months in feces and secretions in the environment and human get infection by inhaling

air borne infectious particles (181).

Avian chlamydiosis has been reported from 471 avian species but mainly from

psittacine birds (33, 94). Human infections are typically acquired from exposure to pet

psittacine birds, however, infection from poultry, free ranging birds have also been

reported (9, 113, 127, 197). The morbidity and mortality among the infected populations is

attributed to many factors. In human psittacosis, a wide spectrum of illness is possibly

ranging from asymptomatic infection to mild influenza-like illness to a fulminate disease

with involvement of several extra pulmonary sites (67, 181).

Attempts have been made in past to serotype or biotype C. psittaci strains of avian

origin by using polyclonal sera, plaque reduction neutralization, microimmunofluroscence

CHAPTER-II

37

and examining inclusion morphology (11, 183). Biological difference has also been

observed among various C. psittaci strains. Two different nucleic acid precursor utilization

patterns and variation in susceptibility to sulfonamides and 5-fluorouridine were observed

(125). Winsor and Grimes also divided 17 C. psittaci strains into low infective and high

infective groups on the basis of infectivity and cytopathology to L929 cells (219). These

biological differences are also well supported by later genetic studies based on gene

specific or whole genome restriction endonuclease analysis and DNA-DNA hybridization

(3, 6, 51, 126, 200).

After the reclassification of order Chlamydiales on the basis of the 16S rRNA gene,

all the avian and 2 mammalian origin strains of the old Chlamydia psittaci group are now

classified as Chlamydophila psittaci (43). Alternatively, ompA gene that encodes major

outer membrane protein (MOMP), which is immuno dominant, is used for serotyping and

genotyping of the C. psittaci strains in epidemiological studies. Various C. psittaci strains

are classified into 8 serovars from A to H using MOMP specific monoclonal antibodies

(Mab) (4, 56, 206, 207) and into 7 genotypes on the basis of the AluI restriction fragment

length polymorphism (RFLP) of the ompA gene (60, 174, 207). But the recent

epidemiological studies and some previous reports have shown the existence of genetically

diverse strains among avian fauna (33, 51, 79, 191, 205). The phylogenetic positions of

these strains have not been established yet. Though the host specificity of different C.

psittaci serotypes and genotypes has been reported (7), but their prevalence across many

avian/mammalian species has been detected (33, 174, 207). Therefore, it is imperative to

genetically characterize all the genetically variant C. psittaci strains irrespective of host

species and to design a suitable and more accommodative classification scheme. Further

identification of C. psittaci strains with higher potential for transmitting and causing

infection of human and other mammalian livestock is also needed.

38

Therefore, in this chapter, diverse C. psittaci strains detected from various avian

and mammalian hosts including human beings were genetically analyzed in ompA gene

locus to know the phylogenetic relationships, pathobiotic implications and for improved

strain categorization. C. psittaci strains with the histories of causing human psittacosis are

either isolated/detected directly from clinically sick patients or from in-contact avian hosts.

Phylogenetic positions of highly diverse strains were confirmed by additionally analyzing

the16S rRNA gene. It was observed that all the genetically diverse C. psittaci strains could

be grouped into 4 broad lineages and 8 small genetic clusters, which encompass all the 7

known and 4 new genotypes as well as all 8 known serotypes. The majority of C. psittaci

strains responsible for the human infection belongs to genetic cluster I.


Chlamydial species and strains: The C. psittaci strains used for genetic analysis

are listed in the Table 7 along with brief historical background. The C. psittaci strains with

the history of human psittacosis includes those isolated in Japan in last 40 years and those

available in DNA data banks.

Chlamydial culture and purification of EBs: McCoy cell lines were used for

primary isolation and multiplication of new and laboratory strains of C. psittaci. The cells

were maintained in Eagle’s minimum essential medium-1 (MEM-1) (Nissui, Japan)

containing 5% fetal bovine serum (FBS), 10 µg/ml gentamicin and 1 µg/ml of

cycloheximide. The EBs were harvested 3 to 4 days after inoculation. Chlamydial EBs

were partially purified as reported by Tamura and Higashi (194). Briefly, harvested cell

suspension was centrifugation at 1,000 ! g for 10 min at 4°C to remove cell debris. The

supernatant was then again centrifuged at 12,000 ! g for 60 minutes at 4°C. The pellets

39

Table 7. C. psittaci strains used to study ompA gene.

Strains Source Human Geographical Reference Data bank

(sample type) infection location accession no.

N-1 Budgerigar (fecal swab) Psittacosisa Japan This study AB284055

Itoh Human (blood and sputum) Psittacosisb Japan This study

c AB284056

Izawa-1 Budgerigar (feces) Psittacosisa Japan This study

d AB284057

Mat116 Chestnut-fronted Macaw (feces) Psittacosish Japan This study AB284058

CP0315 Cockatiel (feces) Psittacosisa Japan This study AB284059

Nosé Budgerigar (feces) Psittacosisa Japan This study

d AB284060

30A Human (ear swab Psittacosisb Japan This study

c, d AB284061

KKCP1 Human (nasopharyngeal swab) Psittacosisb Japan This study

d AB284062

KKCP2 Human (bronchoalveolar fluid) Psittacosisb Japan This study

d AB284063

CPX0308 Oriental White Stork (feces) Unknown Japan This study AB284064

Daruma Parakeet (viscera) Unknown India This studyg AB284065

Borg Human (lung) Pneumonitisb USA This study

e AB284066

6BC Parakeet Unknownf USA (42) M73035

MN Zhang Human/ferret Psittacosisb USA (49) AF269281

CP3 Pigeon Unknown USA (145) AF269265

TT3 Turkey Psittacosis h USA (144) AF269267

NJ1 Turkey Psittacosish USA (142) AF269266

MN/Cal10 Human/ferret Psittacosisb

USA (49) AF269262

MNOs Ostrich Unknown USA (5) AF269264

MNRh Rhea Unknown USA (5) AF269263

WC Bovine (intestinal tissues) Psittacosish

USA (143) AF269269

VS225 Orange-fronted Parakeet Unknown USA (24) AF269261

CT1 Turkey Unknown USA (141) AF269260

41A12 Wild turkey Unknown Belgium (60) AY762609

7778B15 Wild turkey Unknown Belgium (60) AY762612

90/1051 Amazon parrot Unknown Belgium (60) AY762608

92-1293 Turkey Unknown Belgium (208) Y16562

84-55 Parakeet Unknown Belgium (208) CPS16561

98AV2129 Swine Unknown Belgium (211) AY327465

84/2334 Amazon parrot (lung) Unknown Germany (205) AJ310735

GD Duck Unknown Germany (88) AF269261

WS/RT/E30 Duck Unknown Germany (60) AY762613

A22/M Sheep Unknown UK (147) M36703

N352 Duck Unknown UK (157) L04980

Avian type C Unknown avian sp. Unknown UK Storey (1994)j L25436

GV Goat (vaginal sample) Unknown UK Storey (1994)j L25437

7344/2 Pigeon Unknown Italy (60) AY762610

3759/2 Pigeon Unknown Italy (60) AY762611

M56 Muskrat Unknown Canada (182) AF269268

R54 Brown skua (feces) Unknown Sub Antarctic (79) AJ243525

Island

Fulmar#10 Fulmar (cloacal sample) Unknown Faroe Islands (78) AM050561

05/02 Human Psittacosisi Netherlands Heddema et al. 2005)

j DQ324426

OSV Human (sputum/throat wash) Psittacosisi Netherlands (77) DQ230095

CLV Pigeon Unknown Netherlands (77) DQ230096

Orni Pigeon (feces) Unknown Netherlands (76) DQ435299

CLA Pigeon (feces) Unknown Netherlands (76) DQ267973

a Confirmed by detecting chlamydia specific antibodies in psittacosis patients and isolation from companion birds.

b Isolation from human patients having all the typical clinical signs of psittacosis.

c Itoh strain was isolated by Jo et al. 1962 (90) and 30A strain was isolated by Mukai et al. 1985 (134).

d Isolated and kindly provided by Dr. Akira Matsumoto, Kawasaki School of Medicine, Japan (120).

e Isolated by Olson and Treuting, 1944 (ATCC VR-601) (140).

f DNA similar to 6BC (ATCC VR125) was detected in vaginal sample (81).

g Isolated by Yamashita and Hirai, 1981 (221).

h Doubtful history of psittacosis.

i Confirmed by C. psittaci DNA detection in clinical samples of human psittacosis patients.

j Unpublished work.

40

were then resuspended in 0.01M Tris-HCl, pH 7.4, in appropriate volumes and each strain

was stored at -80°C in aliquots. These aliquots were later used for DNA extractions.


was used to extract DNA from samples according to the manufacturer’s instructions. In

brief, about 100 µl partially purified EB were suspended in 700 µl of phosphate-buffered

saline (PBS), pH 7.4, were processed for DNA extraction. DNA was finally dissolved in

50 µl of Tris-EDTA buffer, pH 7.4 (100 mM Tris-HCl, pH 7.4 and 10 mM EDTA, pH 8.0)

and stored at –30°C. The ompA and 16S rRNA genes were amplified in overlapping

fragments by PCR using pair of forward and reverse primers as shown in Table 8.

PCR amplification: The PCR was done in 50 µl reaction mixture containing 0.15

µM of each forward and reverse primer, 250 µM of each dATP, dTTP, dGTP, dCTP, 100

µM of Mg2+

in buffer and 2.5 units of TaKaRa Ex-Taq (Takara Bio Inc., Shiga, Japan) and

2.0 to 5.0 µg of template DNA of each strain. The thermo cycling conditions were:

denaturation for 3 min at 94°C, then 35 cycles of denaturation for 30 sec at 94°C,

annealing for 30 sec at a temperature depending on melting temperature of forward and

reverse primer pairs and extension at 72°C for 60 sec, then final extension for 5 min at

72°C and soaking at 4°C.

DNA sequencing: The cloning of PCR amplified products was done as described

in CHAPTER-I, i.e. 3 to 5 clones with expected size DNA insert were taken for

sequencing. The sequences were assembled and edited using Genetyx-Mac/ATSQ 4.2.3

and Genetyx-Mac, version 13.0.6 (SDC, Tokyo, Japan).

Phylogenetic analysis: For phylogenetic analysis, all the available ompA gene

sequences of C. psittaci strains and each representative species of genera Chlamydophila

and Chlamydia were retrieved from DDBJ. The ompA and 16S rRNA gene sequences were

trimmed to the uniform length to included all the known, genetically diverse C. psittaci

41

Table 8. Oligonucleotide primers used to amplify ompA and 16S rRNA genes.

Name Sequence Positionsa Tm Size

(°C)b

(bp)

ompA gene

M/0PF 5` GCAAGTATAAGGAGTTATTGCTTG 3` -275 to -252 54-56 352

M/0PR 5` CCTACAGGCAAGGCTTGTAAG 3` 56-77 57-60

M/1PF 5` CGGCATTATTGTTTGCCGCTAC 3` 20-41 59 390

M/1PR 5` CGAAGCGATCCCAAATGTTTAAGGC 3` 409-385 59-61

M/2PF 5` CGCTTACGGAAGGCATATGCAAGATG 3` 330-355 62 397

M/2PR 5` GTGAATCACAAATTGTGCTGGGCT 3` 726-703 58-60

M/3PF 5` CGTGGAGCTTTATGGGAATGTGGTTG 3` 607-632 62 330

M/3PR 5` GAGCAATGCGGATAGTATCAGCATC 3` 937-913 60-62

M/4PF 5` GGCGTAAACTGGTCAAGAGCAAC 3` 886-908 60-62 387

M/4PR 5` TCCCAGGTTCTGATAGCGGGACAA 1272-1246 61-63

16S rRNA gene

CPR/P1F 5` CTGAGAATTTGATCTTGGTTCAG 3` 5-27 54-56 400

CPR/P1R 5` CGCTTCGTCAGACTTTCGTCCATT 3` 404-381 60

CPR/P2F 5` CGTCTAGGCGGATTGAGAGATTG 3` 291-313 60-62 418

CPR/P2R 5` CGCATTTCACCGCTACACGTGGAAT 3` 708-684 60-62

CPR/P3F 5` GCGTGTAGGCGGAAAGGAAAGTT 3` 585-607 60-62 413

CPR/P3R 5` CCAGGTAAGGTTCTTCGCGTTGCAT 3` 997-973 59-61

CPR/P4F 5` CCGTGTCGTAGCTAACGCGTTAAGT 3` 860-884 60-62 444

CPR/P4R 5` GGCTAGCTTTGAGGATTTGCTCCAT 3` 1403-1279 59-61

CPR/P5F 5` CGAGGATGACGTCAAGTCAGCAT 3` 1191-1213 60-62 367

CPR/P5R 5` TGTCGACAA AGGAGGTGATCCAG 3` 1557-1537 55-57

a Based on ompA gene open reading frame (ORF) of C. psittaci-6BC strain (accession no. X56980) and 16S

rRNA gene of C. psittaci-Prk/GCP-1 strain (D85713). The negative values indicate the nucleotide position

upstream of respective ORF. bAverages of melting temperatures of forward and reverse primer sets were used in the PCR amplifications.

strains. A total of 1,492 bp fragment of 16S rRNA was analyzed.

The analyzed ompA gene sequences include about 898 to 940 bp fragment

extending from 161 bp downstream from starting codon of ompA gene to 1,098 bp

position. It includes all four hyper variable domains (VDs). The combined constant

domains (CDs) of 631 bp and variable domains of 267 to 309 bp were analyzed separately.

Calculation of percent identity matrix and multiple alignments of the trimmed sequences

was done using ClustalX, version 1.83 (198). Phylogenetic analysis was done with

programs in the PHYLIP (version 3.6a3; [http://evolution.genetics.washington.edu/phylip.

42

html]). The distance matrix between species was computed by DNADIST using Jukes-

Cantor model (93) and clustering of lineages was done by NEIGHBOR using neighbor-

joining method (170). The bootstrap values were calculated to evaluate the branching

reliability of trees from a consensus tree constructed by generating 1,000 random data sets

using SEQBOOT (45).

in-silico AluI random fragment length polymorphism (RFLP) map: The

comparative AluI RFLP maps of different C. psittaci strains were generated by using

Genetyx-Mac, version 13.0.6 (SDC, Tokyo, Japan).

RESULTS

We initially analyzed relative evolution of whole ompA gene among all species of

family Chlamydiaceae vis-à-vis species of new C. psittaci group. Then comparative

analysis of constant/conserved domains (CDs) and variable domains (VDs) of all

genetically variable C. psittaci strain was done for genetic clustering and comparison.

Evolution of ompA gene among the members of Chlamydiaceae family: The

neighbor-joining (NJ) tree in Fig. 6 is showing phylogenetic distances among the

Chlamydophila spp. and Chlamydia spp. infecting human and various types of animal and

avian species. Clear separation of Chlamydophila spp. and Chlamydia spp. into two clans

irrespective of human and animal chlamydiae was observed. Furthermore on the basis of

ompA gene, host specific clustering of Chlamydia spp. or Chlamydophila spp. strains was

observed with exception of C. psittaci strains of avian origin. The C. psittaci strains form

distinct small clusters diverging out from other clusters of Chlamydophila spp.

ompA-gene based genetic clustering of C. psittaci: Various clusters and

subclusters of C. psittaci strains are resolved in the NJ tree of about 940 bp segment of

CPX0308

C. muridarum-MoPn (U60196)C. muridarum-Nigg (AE002160)

C. muridarum-NiggII (X63409)

C. muridarum-SFPD (L19221)

C. trachomatis-D/IC-Cal-8 (X62920)

C. trachomatis-L1(M36533)

C. trachomatis-alfa/95 (U80075)C. trachomatis-LGV (L35605)

C. trachomatis-L-2 (L2/434/Bu) (M14738)

C. trachomatis-E/Bour (DQ064286)

C. trachomatis- IC-Cal3 (X52080)

C. trachomatis-J/UW36/Cx (AF063202)

C. trachomatis- K/UW31/Cx (AF063204)

C. trachomatis- C/TW3/OT (AF352789)

C. suis- PCLH197 (AJ440241)

C. psittaci-BorgC. psittaci-TT3

C. psittaci-Daruma

C. psittaci-Avian Type CC. psittaci-CT1C. psittaci-GDC. psittaci-M56

C. psittaci-MAT116

C. psittaci-30AC. psittaci-A22/M

C. psittaci-N352

C. psittaci-KKCP2C. psittaci-KKCP1

C. psittaci-CP3

C. psittaci-6BC

C. psittaci-MNZhangC. psittaci-N1

C. psittaci-Izawa

C. psittaci-GV

C. psittaci-NoséC. psittaci-Itoh

C. psittaci-CP0315

0.05 units

C. felis-FP Cello (AF269258)C. felis-FP Baker (AF269257)C. felis-FEPN (M73037)

C. felis-FPn/pring (X61096)C. felis-Fe/C56 (AP006861)

C. caviae-GPIC (AE015925)C. caviae-Zhang GPIC

C. caviae-GPIC (AF269282)

C. pecorum-LW613 (AJ440240)

C. pneumoniae-CSF (AF131889)C. pneumoniae-IOL-207 (M64064)C. pneumoniae-AR39 (AE002161)

C. pneumoniae-N16 (L04982)

C. abortus-LLG (AF272945)

C. abortus-B577 (M73036)C. abortus-BA1 (L39020)C. abortus-EBA (AF269256)

C. abortus-S26/3 (X51859)C. abortus-TWC+3/95-H (DQ471955)C. abortus-Taiwan strain (DQ227703)C. abortus-Taiwan strain (DQ478954)C. abortus-OCLH196 (AJ004873)

Genus- Chlamydia

Genus- Chlamydophila

HUMAN

MURINE

GUINEA PIG

RUMINANT

HUMAN

FELINE

SWINE

GUINEA PIG

RUMINANT

EQUINE

AVIAN

AVIAN

AVIAN

Fig. 6. Neighbor joining (NJ) tree of ompA gene ORF showing host species specific clustering of Chlamydophila spp. and Chlamydia spp. infecting human and various animal species. The tree rev--ealed diverse clusters of Chlamydophila spp. of avian origin. The bootstrap values are shown nearto respective nodes. The genetic distance is shown in units scale bar. The accession numbers of C.psittaci strains are shown in Table 7.

100%

100%100%

100% 100%

100%

100%

100%

100% 100%

100%

99.6%

CPX0308

C. caviae-OK135

C. psittaci-WC

C. psittaci -7778B15C. psittaci -VS225

C. psittaci -84/2334C. psittaci -Daruma

C. psittaci -R54C. psittaci -Avian Type CC. psittaci -CT1C. psittaci -GD

C. psittaci -TT3C. psittaci -92-1293C. psittaci -7344/2C. psittaci -NJ1C. psittaci -Borg*

C. psittaci -M56C. psittaci -Mat116

C. psittaci -A22/MC. psittaci -3759/2C. psittaci -MN*C. psittaci -MNOsC. psittaci -MNRhC. psittaci -30A*C. psittaci -N352C. psittaci -98AV2129C. psittaci -WS/RT/E30C. psittaci -CP3

C. psittaci -41A12C. psittaci -KKCP2*C. psittaci -KKCP1*

C. psittaci -6BCC. psittaci -Fulmar#10C. psittaci -MN Zhang*

C. psittaci -84-55C. psittaci -N1*C. psittaci -Nosé*C. psittaci -Itoh*C. psittaci -GVC. psittaci -CP0315*C. psittaci -Izawa-1*C. psittaci -90/1051

0.02 units

C. abortus-B577 (M73036)C. abortus-BA1 (L39020)C. abortus-EBA (AF269256)C. abortus-S26/3 (X51859)

C. abortus-pm112 (AJ005613)C. abortus-pm225 (AJ005617)


C. abortus-pmSH1 (AJ005618)C. abortus-pmd623 (AJ005615)

C. abortus-LW508 (M73040)

C. abortus-TWC+3/95-H (DQ471955)C. abortus-Taiwan strain (DQ227703)

C. abortus-Taiwan strain (DQ478954)

C. abortus-OCLH196 (AJ004873)C. abortus-LLG (AF272945)

C. caviae-GPIC (AE015925)

C. caviae-Zhang GPICC. caviae-GPIC (AF269282)

C. felis-FP Cello (AF269258)C. felis-FP Baker (AF269257)C. felis-FEPN (M73037)C. felis-FPn/pring (X61096)

C. felis-Fe/C56 (AP006861)

C. pecorum-L71 (AF269280)C. pecorum-LW613 (AJ440240)

C. pecorum-1710S (AF269279)

C. pneumoniae-CSF (AF131889)C. pneumoniae-IOL-207 (M64064)C. pneumoniae-AR39 (AE002161)


A

B

E

B/E

?

??

D

F

C

??

?

?

Fig. 7. The partial ompA gene (including VDs) based NJ tree showing the relative evolutionarydistance among different genetic clusters of C. psittaci strains. The known genotypes from A to F and E/B are demarcated by the dotted lines. The unknown/unclassified genotypes are questi--on marked (?). Asterisk mark (*) indicates the C. psittaci strains with human psittacosis history.The bootstrap values are shown againt each node. The genetic distance is shown in units scale bar.

100%

100%

100%

99.2%

86.8%

97.3%

96.7%

95.9%

100%

100%

100%

100%

100%79.4%

100%

100%

100%

100%

100%

45

ompA gene that includes all 4 variable domains (Fig. 7). The distribution analysis of the

genetic distances and percent dissimilarities in C. psittaci strains (conserved regions only)

showed 4 groups of peaks. The ranges of genetic distance values for these 4 groups are

0.002 to 0.04, 0.061to 0.122, 0.133 to 0.156 and 0.194 to 0.211 (Fig. 8).

Fig. 8. Distribution of the genetic distances in the conserved regions of ompA gene of C. psittaci

strains. Genetic distances were calculated by Jukes-Cantor method (93).

Then by tabulating the genetic distance and dissimilarity matrices and plotting NJ

distance trees did the detailed analysis of clustering patterns of C. psittaci strains.

(i) Constant domains. All 21 C. psittaci strains with variant CDs were included in

analysis along with other representative Chlamydophila spp. Previously unclassified

Mat116, R54, Daruma, 84/2334 and CPX0308 strains were observed to be

phylogenetically diverse. On the basis of genetic distance and dissimilarity matrices and

46

the NJ tree of CDs, all the C. psittaci strains were divided into 4 broad lineages and 8

genetic clusters named from I to VIII (Fig. 9-A, Tables 9 and 10). The genetic distance

among strains within a lineage is less than 0.1 (dissimilarity less than 10%). Whereas,

within these lineages strains having genetic distance less than 0.05 (dissimilarity less than

5%) are divided into genetic clusters. Lineage-1 has only one large cluster of genetically

similar strains mainly detected from psittacine bird, pigeons and ratites. The lineage-2 has

5 genetic clusters of genetically diverse strains mostly detected from poultry, water birds,

psittacine and other non-psittacine birds. The lineage-3 is represented by WC strain, a

bovine enteritis isolate, and lineage-4 by CPX0308 strain, detected from an oriental white

stork. The genetic distance among all C. psittaci strains is less than 0.156 except CPX0308

strain that has genetic resemblance to mammalian Chlamydophila spp. than avian type.

The nearest avian strain is V225 with genetic distance of 0.188. The relative difference in

nucleotide and amino acid numbers among 4 lineages and 8 genetic clusters are shown in

Table 10. It showed that seemingly large number of nucleotide variations do not reflect the

resultant amino acid variation within respective lineages or genetic clusters.

(ii) Variable domains. Total 18 C. psittaci strains having variation in VDs regions

were analyzed. KKCP1, KKCP2, Mat156, R54, Daruma, 84/2334 and CPX0308 strains

were observed to be phylogenetically distinct (Fig. 9-B). All the 8 genetic clusters from I

to VIII were also distinguishable but lower degree of percent identity and higher genetic

distance was found within each genetic cluster and designated lineages (Fig. 9-B and Table

11). The percent identity between 4 lineages was found to be less than 67%. Each genetic

cluster contains the same type of strains as that in CDs based tree.

Phylogenetic positions of CPX0308 and Daruma strain: Two highly genetically

diverse strains of avian origin CPX0308 and Daruma were compared with other members

of family Chlamydiaceae. The NJ tree of 16S rRNA gene is shown in Fig. 10. CPX0308

47

Fig. 9. NJ trees based on the constant domain regions (A) and the variable domains (B), of

ompA gene showing genetic clusters of C. psittaci strains and other Chlamydophila spp.

The genetic clusters of C. psittaci strains from I to VIII are demarcated by the vertical solid

lines and known genotypes from A to F and E/B by dotted lines. The unknown/unclassified

genotypes are question marked (?). The C. psittaci strains with the history of human

psittacosis are marked by asterisk (*). The genetic distance is shown in units scale bar.

(A) (B)

Izawa-1*CP0315*

84-5590/1051Itoh

N1NoséMN Zhang*

6BCFulmar#10

41A12CP3

N352WS/RT/E30

MNRh

MNOsMN

KKCP1KKCP2

A22/M3759/2

98AV2129

30AMat116

M56WC

92-12937344/2BorgNJ1TT3

7778B15VS225

Avian type CCT1GD

R5484/2334

Daruma

CPX0308

0.05 units

C. abortus-B577 (M73036)C. abortus-BA1 (L39020)C. abortus-EBA (AF269256)C. abortus-S26/3 (X51859)



C. abortus-pmSH1 (AJ005618)C. abortus-pmd623 (AJ005615)

C. abortus-LW508 (M73040)C. abortus-TWC+3/95-H (DQ471955)

C. abortus-Taiwan strain (DQ227703)C. abortus-Taiwan strain (DQ478954)

C. abortus-OCLH196 (AJ004873)C. abortus-LLG (AF272945)



C. felis-FP Cello (AF269258)

C. felis-P Baker (AF269257)C. felis-FEPN (M73037)

C. felis-FPn/pring (X61096)C. felis-Fe/C56 (AP006861)

C. pecorum-L71 (AF269280)C. pecorum-LW613 (AJ440240)

C. pecorum-1710S (AF269279)C. pneumoniae-CSF (AF131889)C. pneumoniae-IOL-207 (M64064)C. pneumoniae-AR39 (AE002161)


C. caviae-GPIC-Zhang

GV

I

VII

II

III

VI

V

IV

VIII

A

B

E

B/E

??

?

D

F

C

?

?

?

**

*

*

*

*

**

6BC

84-55

MN Zhang*Izawa-1*

GVFulmar#10

CP0315*90/1051

Itoh*

Nosé*

05/02*

41A12

KKCP1*KKCP2*

CP3

98AV2129N352WS/RT/E30

30A*3759/2A22/MMNOs

MNRhMN*

Mat116M56

WC

92-12937344/2Borg*NJ1TT3

Avian Type CCT1GD

R547778B15VS225

84/2334Daruma

CPX0308

C. caviae-GPIC

0.05 units

C. abortus-B577 (M73036)C. abortus-BA1 (L39020)C. abortus-EBA (AF269256)

C. abortus-S26/3 (X51859)C. abortus-pm112 (AJ005613)

C. abortus-pm225 (AJ005617)C. abortus-pm234 (AJ004874)C. abortus-pm326 (AJ004875)C. abortus-pmSH1 (AJ005618)

C. abortus-pmd623 (AJ005615)

C. abortus-LW508 (M73040)

C. abortus-TWC+3/95-H (DQ471955)

C. abortus-Taiwan strain (DQ227703)C. abortus-Taiwan strain (DQ478954)

C. abortus-OCLH196 (AJ004873)

C. abortus-LLG (AF272945)



C. felis-FP Cello (AF269258)

C. felis-FP Baker (AF269257)C. felis-FEPN (M73037)

C. felis-FPn/pring (X61096)

C. felis-C56 (AP006861)

C. pecorum-L71 (AF269280)C. pecorum-LW613 (AJ440240)C. pecorum-1710S (AF269279)

C. pneumoniae-CSF (AF131889)C. pneumoniae-IOL-207 (M64064)

C. pneumoniae-AR39 (AE002161)


N1*

I

VII

II

III

VI

V

IV

VIII

A

B

E

B/E

?

??

D

F

C

?

?

?

?

100%

100%

100%

100%

100%

93.7%

84.6%

97.9%

100%

91.2%

100%

100%

100%

100%

100%

100%

99.6%

99.4%

100%

100%

100%

99.2%

100%

100%

94.9%

92.4%

82%

100%

100%

100%

92.9%

Table 9. Grouping of genetically diverse C. psittaci strains based on the genetic distance in constant domains of the ompA gene, calculated by Jukes-Cantor method (93). The C. psittaci strains having genetic

distance less than 0.1 are grouped in a lineage and those less than 0.05 genetic distance are grouped togather into a single cluster. Only the representative genetically variant strains of C. psittaci were taken for

analysis. The other Chlamydophila species are shown with grey background. Different genetic clusters are demarcated by solid lines and lineages by dotted lines.

Lin.-3 Lin.-4

Chlamydial species Accession 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

no.

1-C. psittaci-GV L25437 0.000

2-C. psittaci-Nosé AB284060 0.002 0.000

3-C. psittaci-Izawa-1 AB284057 0.003 0.002 0.000

4-C. psittaci-6BC M73035 0.003 0.002 0.003 0.000

5-C. psittaci-MN Zhang AF269281 0.005 0.003 0.005 0.005 0.000

6-C. psittaci-41A12 AY762609 0.006 0.005 0.006 0.003 0.008 0.000

7-C. psittaci-MN AF269262 0.006 0.005 0.006 0.003 0.008 0.003 0.000

8-C. psittaci-KKCP1 AB284062 0.006 0.005 0.006 0.003 0.008 0.003 0.000 0.000

9-C. psittaci-CP3 AF269265 0.010 0.008 0.010 0.006 0.011 0.003 0.006 0.006 0.000

10-C. psittaci-8455 CPS16561 0.013 0.011 0.013 0.013 0.014 0.016 0.016 0.016 0.019 0.000

11-C. psittaci-Mat116 AB284058 0.019 0.018 0.019 0.016 0.021 0.019 0.019 0.019 0.023 0.029 0.000

12-C. psittaci-M56 AF269268 0.034 0.032 0.034 0.031 0.036 0.034 0.034 0.034 0.031 0.044 0.031 0.000

13-C. psittaci-Borg AB284066 0.116 0.114 0.116 0.113 0.114 0.116 0.116 0.116 0.120 0.124 0.129 0.118 0.000

14-C. psittaci-92-1293 Y16562 0.118 0.116 0.118 0.114 0.116 0.118 0.118 0.118 0.122 0.126 0.131 0.120 0.002 0.000

15-C. psittaci-7778B15 AY762612 0.137 0.135 0.137 0.133 0.135 0.137 0.137 0.137 0.141 0.145 0.141 0.150 0.071 0.073 0.000

16-C. psittaci-VS225 AF269261 0.139 0.137 0.139 0.135 0.137 0.139 0.139 0.139 0.139 0.147 0.143 0.148 0.073 0.075 0.002 0.000

17-C. psittaci-Daruma AB284065 0.139 0.137 0.139 0.135 0.137 0.139 0.139 0.139 0.143 0.147 0.139 0.143 0.082 0.084 0.080 0.082 0.000

18-C. psittaci-84/2334 AJ310735 0.141 0.139 0.141 0.137 0.139 0.141 0.141 0.141 0.145 0.148 0.141 0.145 0.084 0.086 0.082 0.084 0.002 0.000

19-C. psittaci-R54 AJ243525 0.145 0.143 0.145 0.141 0.143 0.145 0.145 0.145 0.148 0.152 0.141 0.145 0.077 0.078 0.063 0.065 0.051 0.053 0.000

20-C. psittaci-GD AF269261 0.150 0.148 0.150 0.147 0.148 0.150 0.150 0.150 0.154 0.158 0.147 0.145 0.073 0.075 0.061 0.063 0.070 0.071 0.058 0.000

21-C. psittaci-WC AF269269 0.148 0.147 0.148 0.145 0.145 0.148 0.148 0.148 0.152 0.156 0.154 0.152 0.105 0.107 0.113 0.114 0.114 0.116 0.128 0.120 0.000

22-C. psittaci-CPX0308 AB284064 0.211 0.209 0.211 0.207 0.211 0.205 0.207 0.207 0.205 0.209 0.196 0.205 0.196 0.198 0.190 0.188 0.207 0.207 0.205 0.194 0.192 0.000

23-C. abortus-S26/3 X51859 0.152 0.150 0.152 0.148 0.150 0.152 0.152 0.152 0.156 0.160 0.148 0.148 0.091 0.093 0.071 0.073 0.087 0.089 0.065 0.080 0.128 0.203 0.000

24-C. caviae-GPIC AE015925 0.176 0.174 0.176 0.172 0.174 0.176 0.176 0.176 0.180 0.184 0.166 0.170 0.164 0.166 0.170 0.172 0.182 0.184 0.174 0.172 0.166 0.174 0.174 0.000

25-C. felis-C56 AP006861 0.188 0.186 0.188 0.184 0.182 0.188 0.188 0.188 0.192 0.200 0.180 0.180 0.160 0.162 0.160 0.162 0.172 0.174 0.154 0.166 0.174 0.186 0.168 0.170 0.000

26-C. pneumoniae-AR39 AE002161 0.284 0.282 0.284 0.280 0.282 0.280 0.280 0.280 0.280 0.296 0.273 0.289 0.280 0.282 0.284 0.282 0.293 0.293 0.296 0.289 0.282 0.312 0.305 0.301 0.277 0.000

27-C. pecorum-L71 AF269280 0.313 0.311 0.313 0.308 0.313 0.308 0.308 0.308 0.313 0.313 0.316 0.316 0.289 0.292 0.304 0.306 0.316 0.318 0.311 0.311 0.308 0.320 0.320 0.304 0.333 0.269 0.000

Designated

genetic clusters VIII Other Chlamydophila spp.III IV V VIII II

Lineage-1 Lineage-2

VI

Table 10. The difference in number of nucleotides (upper right triangular half) and amino acids (lower triangular half) among different lineages and genetic clusters of C. psittaci strains in constant domains of

the ompA gene. Only the representative genetically variant strains of C. psittaci were taken for analysis. Other Chlamydophila species are demarkated by grey background. Different genetic clusters are

demarcated by solid lines and lineages by dotted lines.

Lin.-3 Lin.-4

Chlamydial species Accession 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

no.

1- C. psittaci- GV L25437 0 1 2 2 3 4 4 4 6 8 12 21 68 69 79 80 80 81 83 86 85 116 87 99 105 150 163

2- C. psittaci- Nosé AB284060 1 0 1 1 2 3 3 3 5 7 11 20 67 68 78 79 79 80 82 85 84 115 86 98 104 149 162

3- C. psittaci- Izawa-1 AB284057 2 1 0 2 3 4 4 4 6 8 12 21 68 69 79 80 80 81 83 86 85 116 87 99 105 150 163

4- C. psittaci- 6BC M73035 2 1 2 0 3 2 2 2 4 8 10 19 66 67 77 78 78 79 81 84 83 114 85 97 103 148 161

5- C. psittaci- MN Zhang AF269281 1 0 1 1 0 5 5 5 7 9 13 22 67 68 78 79 79 80 82 85 83 116 86 98 102 148 163

6- C. psittaci- 41A12 AY762609 4 3 4 2 3 0 2 2 2 10 12 21 68 69 79 80 80 81 83 86 85 113 87 99 105 148 161

7- C. psittaci- MN AF269262 4 3 4 2 3 1 0 0 4 10 12 21 68 69 79 80 80 81 83 86 85 114 87 99 105 148 161

8- C. psittaci- KKCP1 AB284062 4 3 4 2 3 1 0 0 4 10 12 21 68 69 79 80 80 81 83 86 85 114 87 99 105 148 161

9- C. psittaci- CP3 AF269265 5 4 5 3 4 1 2 2 0 12 14 19 70 71 81 80 82 83 85 88 87 113 89 101 107 147 169

10- C. psittaci- 84-55 CPS16561 4 3 4 4 3 6 6 6 7 0 27 18 72 73 83 84 84 85 87 90 89 115 91 103 111 155 163

11- C. psittaci- Mat116 AB284058 4 3 4 2 3 4 4 4 5 6 0 19 75 76 81 82 80 81 81 84 88 109 85 94 101 145 164

12- C. psittaci- M56 AF269268 4 3 4 2 3 4 4 4 3 6 4 0 69 70 86 85 82 83 83 83 87 113 85 96 101 152 164

13- C. psittaci- Borg AB284066 10 9 10 8 9 10 10 10 11 12 10 10 0 1 43 44 49 50 46 44 62 109 54 93 91 148 153

14- C. psittaci- 92-1293 Y16562 11 10 11 9 10 11 11 11 12 13 11 11 1 0 44 45 50 51 47 45 63 110 55 94 92 149 154

15- C. psittaci- 7778B15 AY762612 13 12 13 11 12 13 13 13 14 15 11 13 7 8 0 1 48 49 38 37 66 106 43 96 91 150 159

16- C. psittaci- VS225 AF269261 13 12 13 11 12 13 13 13 14 15 11 13 7 8 0 0 49 50 39 38 67 105 44 97 92 149 160

17- C. psittaci- Daruma AB284065 9 8 9 7 8 9 9 9 10 11 9 9 3 4 4 4 0 1 31 42 67 114 52 102 97 154 162

18- C. psittaci- 84/2334 AJ310735 10 9 10 8 9 10 10 10 11 12 10 10 4 5 5 5 1 0 32 43 68 114 53 103 98 154 163

19- C. psittaci- R54 AJ243525 10 9 10 8 9 10 10 10 11 12 9 10 4 5 4 4 1 2 0 35 74 107 74 94 98 145 161

20- C. psittaci- GD AF269261 11 10 11 9 10 11 11 11 12 13 9 11 6 7 6 6 4 5 4 0 70 113 39 98 88 155 160

21- C. psittaci- WC AF269269 12 11 12 10 11 11 11 11 12 14 12 12 7 8 10 10 8 9 9 20 0 108 48 97 94 152 162

22- C. psittaci- CPX0308 AB284064 31 30 31 29 30 28 28 28 29 33 29 31 27 28 26 26 28 28 28 29 27 0 112 98 104 161 165

23- C. abortus-S26/3 X51859 12 11 12 10 11 12 12 12 13 14 11 12 8 9 5 5 5 6 4 8 9 27 0 98 95 159 166

24- C. caviae- GPIC AE015925 20 19 20 18 19 20 20 20 21 22 16 20 19 20 15 15 18 19 18 20 19 28 18 0 96 158 157

25- C. felis-C56 AP006861 21 20 21 19 20 21 21 21 22 23 19 21 16 17 18 18 16 17 17 19 18 27 18 22 0 146 163

26- C. pneumoniae-AR39 AE002161 45 44 45 44 44 44 44 44 45 47 42 46 43 44 41 41 41 41 41 42 42 42 42 42 39 0 145

27- C. pecorum-L71 AF269280 44 43 44 43 43 43 43 43 44 46 44 45 43 44 44 44 41 42 41 43 43 46 43 47 45 38 0

Other Chlamydophila spp.II III IV V VI VII VIII

Lineage-1 Lineage-2

IDesignated genetic clusters

Table 11. Percentage identity matrix (PIM) of nucleotides in the genetically variable regions (VDs) of ompA gene of different lineages and genetic clusters of C. psttaci strains. The

PIM was calculated by ClustalX, version 1.83. The other Chlamydophila species are marked by grey background. The lineages are marked by dotted lines, whereas, the genetic

clusters are marked by solid lines.

Lin.-3 Lin.-4

Chlamydial species Accession No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

1-C. psittaci-05/02 DQ324426 100

2-C. psittaci- Nosé AB284060 100 100

3-C. psittaci- 84-55 CPS16561 99 100 100

4-C. psittaci- 6BC M73035 99 100 99 100

5-C. psittaci- CP3 AF269265 98 98 98 98 100

6-C. psittaci- KKCP1 AB284062 97 98 97 98 100 100

7-C. psittaci- MN AF269262 97 97 97 98 99 98 100

8-C. psittaci- WS/RT/E30 AY762613 97 97 97 98 99 98 99 100

9-C. psittaci- Mat116 AB284058 95 95 95 95 95 94 94 94 100

10-C. psittaci- M56 AF269268 89 89 88 89 87 87 87 87 89 100

11-C. psittaci- Borg AB284066 66 66 67 66 66 66 66 66 66 67 100

12-C. psittaci- VS225 AF269261 60 61 61 61 61 61 60 61 60 60 72 100

13-C. psittaci- R54 AJ243525 60 59 60 59 59 59 59 59 59 60 71 84 100

14-C. psittaci- 84/2334 AJ310735 56 56 56 56 56 56 56 56 56 56 67 83 81 100

15-C. psittaci- Daruma AB284065 56 56 57 56 57 56 57 56 57 57 68 85 81 98 100

16-C. psittaci- GD AF269261 55 55 55 55 55 55 55 55 54 56 70 76 79 77 78 100

17-C. psittaci- WC AF269269 62 61 62 61 61 61 61 61 60 61 70 65 66 65 65 63 100

18-C. psittaci- CPX0308 AB284064 59 58 59 58 58 58 58 58 58 60 67 67 62 60 61 63 62 100

19-C. abortus-S26/3 X51859 59 59 60 59 59 59 58 59 58 60 68 89 82 82 85 73 64 65 100

20-C. felis-C56 AP006861 59 59 59 59 59 60 59 59 59 59 68 69 72 63 65 64 62 62 69 100

21-C. pneumoniae-AR39 AE002161 55 55 55 55 56 55 56 56 56 56 60 61 58 56 58 59 54 62 61 59 100

22-C. caviae- GPIC AE015925 54 54 54 54 54 55 55 55 55 53 62 69 70 65 67 64 61 59 67 67 58 100

23-C. pecorum-L71 AF269280 52 52 52 52 52 52 52 52 52 52 57 57 57 57 57 53 55 60 56 58 53 61 100

VII VIII Other Chlamydophila spp.

Lineage-2

III IV V VIDesignated genetic clusters I

Lineage-1

II

C. psittaci-Koala (AB001789)C. psittaci-Sugimoto-F (AB001812)C. psittaci-Mizuno-1F (AB001790)C. psittaci-Izawa-1 (AB001788)C. psittaci-Itoh (AB001787)C. psittaci-GCP-1 (AB001786)C. psittaci-Bud-5695 (AB001782)C. psittaci-6BC (CPU68447)

C. psittaci-Ohmiya (AB001791)C. psittaci-Sugawara (AB001813)C. psittaci-Bud-1 (AB001779)

C. psittaci-Bud-11F (AB001780)C. psittaci-Bud-16F (AB001781)

C. psittaci-P1307 (AB001795)C. psittaci-P1321 (AB001798)

C. psittaci-P1313 (AB001796)C. psittaci-P1315 (AB001797)C. psittaci-P1605 (AB001799)C. psittaci-T4 (AB001815)C. psittaci-PCM27 (AB001804)C. psittaci-PCM30 (AB001805)

C. psittaci-Cal-10 (AB001784)C. psittaci-P1015 (AB001792)C. psittaci-P1041 (AB001793)C. psittaci-P1305 (AB001794)C. psittaci-P1646 (AB001800)C. psittaci-P1888 (AB001801)C. psittaci-PCM131 (AB001803)C. psittaci-Tk-Cal (AB001816)

C. psittaci-PCM9 (AB001808)C. psittaci-PCM44 (AB001806)C. psittaci-PCM55 (AB001807)

C. psittaci-Frt-Hu/Ca110 (D85712)C. psittaci-PgAu46 (AB001802)C. psittaci-T3 (AB001814)

C. psittaci-Tk-NJ (CPU68419)C. psittaci-Hu/Borg (D85711)

C. psittaci-Prk46 (AB001809)C. psittaci-Prk48 (AB001810)C. psittaci-Daruma (AF481048)C. abortus-Ov/B577 (D85709)

C. abortus-EBA (U76710)C. abortus-S26/3 (CR848038)

C. psittaci-Prk49 (AB001811)C. caviae-GPIC (AE015925)

C. caviae-OK135C. caviae- GPIC (D85708)

CPX0308 (AB285329)C. felis-Fe/145 (D85702)

C. felis-Fe/C56 (AP006861)C. felis-Fe/429 (D85707)

C. felis-Cello (D85706)C. felis-Fe/Pn-1(D85701)

C. felis- Fe/164 (D85704)C. pecorum-Bo-1485 (AB001774)

C. pecorum-Bo-Yokohama (AB001776)C. pecorum-Bo/Shizuoka (D85714)

C. pecorum-Koala type II (D85717)C. pneumoniae-CWL029 (AE001668)

C. pneumoniae-TW183 (AE017160)

0.005 units

Fig. 10. The 16S rRNA gene based neighbor-joining (NJ) tree showing the evolutionary distanceof the CPX0308 and Daruma strains of C. psittaci (bold letter) in comparison to the other known species of genus Chlamydophila. The accession numbers are shown in parentheses. The geneticdistance is shown in units scale bar. The bootstrap values are shown againt nodes.

73.4%

77.3%

84.8%

79.4%

94.8%

98.7%80.6%

100%

99.8%

100%

100%

53

strain showed genetic distance of 0.006 from the closest relative, Daruma strain, while

Daruma strain was genetically closest, that is 0.001 to C. abortus S26/3 strain, (Table 12-

A). According to the CDs of the ompA gene, CPX0308 strain was the closest to C. caviae

GPIC strain with genetic distance of 0.176, while Daruma strain showed genetic distance

of 0.051 from R54 strains of C. psittaci. However, on the basis of partial ompA gene

(including VDs), the closest relatives of CPX0308 and Daruma were V225 and R54 strains

of C. psittaci with distance of 0.255 and 0.096, respectively (Table 12-B and C).

Therefore, Daruma strain was phylogenetically intermediate between the clusters formed

by C. psittaci and C. abortus species, whereas, CPX0308 strain appeared to be

intermediate between cluster of C. psittaci and C. abortus species and that of C. caviae and

C. felis species. Therefore, CPX0308 and Daruma strains are grouped into the lineage-4

(cluster VIII) and lineage-2 (clusters IV), respectively.

Human psittacosis strains: The majority of C. psittaci strains isolated from human

psittacosis patients or from in-contact avian species was observed to be belonging to

lineage-1 and genetic cluster I. These strains are predominantly prevalent among psittacine

and pigeons, though also reported from other avian and mammalian species. The recently

reported OSV and 05/02 strains (Table 7) from human psittacosis outbreak were also

similar to strains of genetic cluster I. OSV strain is 99.78%, 99.02% and 99.78% similar to

6BC, CP3 and MN strains, respectively, whereas, 05/02 strain was 99.80%, 99.09% and

98.88% similar to 6BC, CP3 and MN strains, respectively. Interestingly, KKCP1 and

KKCP2 strains showed characteristics of both MN (formerly grouped as genotype/serotype

E) and CP3 (formerly grouped as genotype/serotype B) and form intermediate subcluster

between them (Fig. 9 B). Second type of C. psittaci strains involved in human psittacosis

belongs to genetic cluster II that has been known as serotype/genotype D strain. These

strains are mostly reported from poultry birds.

Table 12. Genetic distance of CPX0308 and Daruma strains vis-à-vis other Chlamydophila spp. and Chlamydia spp. based on A) 16S rRNA gene, B) constant domains of partial ompA gene, C) partial ompA gene including variable

domains. The distance was calculated by Jukes-Cantor method (93) using Phylip. The two nearest species/strains are shown in grey background.

A)C. psittaci C. psittaci C. psittaci C. psittaci C. psittaci C. psittaci C. psittaci C. psittaci C. psittaci C. psittaci C. psittaci C. psittaci C. psittaci C. abortus C. caviae C. felis C. pecorum C. pneumoniae C. trachomatis C. suis C. muridarum

CPX0308 Daruma 6BC Itoh PCM9 Cal10 P1307 P1015 T3 Borg Tk-NJ PCM27 T4 S26/3 GPIC Fe/C56 Bo-1485 CWL029 D/UW-3/CX S45 Nigg

CPX0308 0.000 0.006 0.007 0.007 0.010 0.009 0.010 0.009 0.010 0.009 0.009 0.010 0.010 0.007 0.009 0.017 0.039 0.046 0.049 0.059 0.045

Daruma 0.006 0.000 0.002 0.002 0.005 0.004 0.005 0.004 0.007 0.006 0.006 0.005 0.005 0.001 0.009 0.016 0.038 0.043 0.051 0.059 0.045

Accession no. AB285329 AF481048 CPU68447 AB001787 AB001808 AB001784 AB001795 AB001792 AB001814 D85711 CPU68419 AB001804 AB001815 CR848038 AE015925 AP006861 AB001774 AE001668 AE001347 U73110 AE002160

B)

C. psittaci C. psittaci C. psittaci C. psittaci C. psittaci C. psittaci C. psittaci C. psittaci C. psittaci C. psittaci C. psittaci C. psittaci C. psittaci C. abortus C. caviae C. felis C. pecorum C. pneumoniae C. trachomatis C. suis C. muridarum

CPX0308 Daruma 6BC Nosé CP3 MN Mat116 M56 R54 Borg GD VS225 WC S26/3 GPIC Fe/C56 LW613 AR39 D/UW-3/CX PCLH197 Nigg

CPX0308 0.000 0.207 0.207 0.209 0.205 0.207 0.196 0.205 0.205 0.196 0.194 0.188 0.192 0.203 0.176 0.186 0.296 0.312 0.322 0.339 0.319

Daruma 0.207 0.000 0.135 0.137 0.143 0.139 0.139 0.143 0.051 0.082 0.070 0.082 0.114 0.087 0.184 0.172 0.311 0.293 0.331 0.317 0.351

C)

CPX0308 0.000 0.288 0.293 0.296 0.294 0.297 0.288 0.293 0.281 0.257 0.271 0.255 0.273 0.268 0.260 0.263 0.385 0.377 0.458 0.461 0.401

Daruma 0.288 0.000 0.239 0.239 0.242 0.239 0.234 0.241 0.096 0.160 0.121 0.108 0.198 0.111 0.252 0.252 0.409 0.373 0.448 0.449 0.424

Accession no. AB284064 AB284065 M73035 AB284060 AF269265 AF269262 AB284058 AF269268 AJ243525 AB284066 AF269261 AF269261 AF269269 X51859 AE015925 AP006861 AJ440240 AE002161 X62920 AJ440241 AE002160

55

Comparison of genetic cluster and AluI RFLP based genotyping method: The

AluI based RFLP maps are shown in Fig. 11. Mat116, KKCP1 and KKCP2 strain have

similar RFLP pattern but are genetically diverse with one another (Fig. 7 and Fig. 9).

Similarly, some closely related strains having single nucleotide polymorphism (SNP) at a

few positions with or without amino acid change could not be resolved by AluI based

RFLP genotyping.

DISCUSSION

For differentiation of C. psittaci strains, currently serotyping and genotyping

methodologies are used which have their own advantages and disadvantages (4, 174, 207).

But due to the incessant reports of existence of C. psittaci strains with major or minor genetic

variation among avian species and human psittacosis cases (33, 60, 77, 191), an alternative

and more accommodative strain classification system is desired. Existing methods are also

reported to have some ambiguity in strain differentiation (191, 207).

Therefore, in this study, we introduce an alternative genetic cluster based strain

classification scheme by using genetic distances and percentage identities. We analyzed the

conserved regions of the ompA gene, which have genetic evolution trajectory similar to other

portion of chlamydial genome (24). The VDs regions with excessive homoplasy were

analyzed separately. All the genetically variable strains are grouped into 4 lineages, which are

further subdivided into 8 genetic clusters named from I to VIII. This scheme accommodates

all C. psittaci strains with major or minor genetic differences and groups them according to

the actual genetic variation.

The genetic cluster I include 13 types of genetically variant C. psittaci strains. It

accommodates all closely related subclusters like that formed by KKCP1 and KKCP2 strain

due to small variations in VD2 and VD4 regions. Similarly a recently known E/B genotype

having only 2-nucleotide variations in VD4 region (60) was clubbed together with other

100 500 1000

CPX0308 (14)

1200

30A, MN, N352 (13)

1200

Nosé, MN Zhang (12)

1200

KKCP1, KKCP2 (11)

Borg, TT3, NJ1 (8)

1200

Mat116 (11)

1200

Daruma(10)

1200

Izawa-1 (12)

1200

6BC (12)

1200

(10)

GD (9)

CP3

CP0305, GV, Itoh

84/2334* (7)

VS225 (12)

(10)WC

Genotypes Genetic

clusters

A

A

A

B

New genotype

New genotype

Unclassified genotype

New genotype

New genotype

E

C

D

F

Strains Restriction

sites no.

Nucleotide positions on ompA gene ORF

100 200 300 400 500 600 700 800 900 1000 1100 1200

I

I

I

I

I

I

I

VI

II

III

IV

IV

VII

VIII

1200

M56 (9) I

(10) VR54*

Fig. 11. RFLP restriction map of the ompA gene based on AluI enzyme used to categorize various C. psittaci strains into A to F genotypes.

The unknown/unclassified genotypes and corresponding genetic clusters ( I to VIII) are also shown. The asterik (*) indicates that ORF of

R54 and 84/2334 strains are incomplete from 3’ and 5’ ends respectively. The variable domains (VDs) are demarcated by thick lines in the

top line diagram of ompA gene, which is showing the nucleotide positions in the ORF.




VD1 VD2 VD3 VD4

57

closely related subclusters. The R54 strain detected from brown skua (79) has been placed

in genetic cluster V in a group of highly diverse strains (lineage-2). Similarly, we grouped

Daruma strain along with 84/2334 strain in genetic cluster IV, which is intermediate in

evolution between C. abortus and C. psittaci groups (205). The 84/2334 strain was

previously classified as serotype A (207) based on Mab against MOMP, which include

genetically different group of strains. The CPX0308 strain has been grouped as genetic

cluster VIII in lineage-4. Hence, proposed scheme of strain classification correctly group

different genetically variant strains.

The PCR based chlamydial detection techniques have overcome the problem

associated with chlamydial isolations for diagnosing the infection. These procedures are

now increasingly used in large-scale epidemiological studies, strain differentiations and for

quick clinical diagnosis. The genetic distance based classification can also accommodates

uncultured chlamydiae. It also overcomes the constrains associated with serotyping

techniques that need isolation of C. psittaci strains before typing, which is not possible in

some cases due to zoonotic risk. Furthermore, it is known that immunodominat epitopes

recognized by monoclonal antibodies used in serotyping studies, reside in the hypervariable

regions of ompA gene. Therefore, the correspondence between ompA-RFLP and MOMP

serotyping is not absolute and the results in some strains can be misinterpreted (174, 191).

The AluI RFLP pattern based ambiguities among Mat116, KKCP1 and KKCP2 strains also

indicate that genotypic classification using only one restriction enzyme is not representative.

Therefore, the new strain classification scheme represents much more the actual genetic

diversity among C. psittaci strains.

The lineages of C. psittaci strains also reflect their evolutionary patterns. The

lineages-1 strains have adapted to avian species and appear mostly capable of infecting in-

contact animals and human beings. The strains of lineage-2 contains 5 genetic clusters of

strains that appeared to be recently adapted to avian species as was estimated by their low

58

prevalence. The C. abortus group that has been recognized as distinct pathobiotype (40,

43, 199), has been evolving from hypothetic ancestor belonging to lineage-2. The

CPX0308 strain analyzed by ompA gene and additional 16S rRNA gene sequences seems

to be an intermediate between group of C. psittaci and C. abortus and other

Chlamydophila spp. of mammalian origins. This CPX0308 strain may have the common

ancestor for this cluster. Further genetic studies of such strains in details are needed to

establish their exact phylogenetic positions in relation to other chlamydial species.

The evolutionary basis of epidemiological success and pathobiology of C. psittaci

strains is difficult to examine due to non-availability of whole genome sequence data. In

our study, the analysis of C. psittaci strains isolated/detected from human psittacosis

patients or in-contact avian hosts revealed that C. psittaci strains belongs to genetic

clusters I are mostly responsible for human psittacosis. Recently, similar types of strains

were reported from an outbreak in Netherlands (77). As these types of strains are mostly

prevalent in psittacine birds, there are numbers of reports of sporadic human psittacosis

cases or large scale outbreaks from ancient time to very recent time, where the probable

source is mentioned as psittacine birds or pigeons, but the genotypes of these strains were

not studied (37, 107, 113, 116, 123, 197). These types of strains are also reported from the

cases of swine, 98AV2129 strain (211), ewes, A22/M strain (147), Moorish tortoise (209)

and dog (68). This reflects the pathogenic potential of this group of strains not only to

human but also to other in-contact mammalian and non-mammalian hosts. The Borg strain,

which is involved in human psittacosis, belongs to genetic cluster II. Similar genotypes

may be involved in human psittacosis cases among the poultry industry workers (8, 117,

127).

Therefore, in this investigation, we grouped the C. psittaci strains according to a

modified scheme, which is genetically accommodative and appropriately represents actual

59

genetic diversity among C. psittaci strains. It may help in understanding the evolutionary

patterns of C. psittaci strains and identifying the C. psittaci pathobiotypes associated with

avian/animals or zoonotic infections.

SUMMARY

Different genetically diverse C. psittaci strains detected from various avian and

mammalian hosts including human beings were genetically analyzed to evaluate the

phylogenetic relationships, pathobiotic implications and to improve the strain

categorization. All of the 11 new C. psittaci strains together with the previously described

35 strains were genetically analyzed in ompA gene locus. Conserved and variable domains

of ompA gene were analyzed separately and 16S rRNA gene of two highly diverse strains

CPX0308 and Daruma was also studied. On the basis of the conserved region of ompA

gene, 4 lineages of C. psittaci strains with genetic distance of less than 0.1 (dissimilarity

less than 10%) were identified. The C. psittaci strains of 4 lineages, with genetic distance

of less than 0.05 (dissimilarity less than 5%) were subdivided into 8 genetic clusters. The

ompA gene loci of the C. psittaci strains of lineage-1 are relatively conserved and the

strains were frequently involved in infection of human (psittacosis) and other mammalian

species. Lineage-2 strains are genetically diverse and divided into 5 genetic clusters.

Lineage-3 and 4, represented by WC and CPX0308 strains, respectively, are genetically

more close to Chlamydophila spp. causing infection among mammalian hosts. This scheme

classifies diverse strains according to their genetic variations and phylogenetic distances,

which is more appropriate to accommodate and classify closely and distantly related C.

psittaci strains. It may be useful to differentiate even uncultured C. psittaci strains detected

in clinical diagnosis.

60

Examination of virulence patterns of the Chlamydophila psittaci strains

predominantly associated with avian chlamydiosis and human psittacosis

using BALB/c mice

INTRODUCTION

The Chlamydophila psittaci causes “avian chlamydiosis” among diverse avian

species, which is characterized by systemic, acute or chronic disease or

subclinical/inapparent infection with intermittent shedding of infectious EBs. Different C.

psittaci strains vary in virulence, which gives rise to variable incubation periods, clinical

signs and recovery time. Typical signs of a clinically apparent infection with virulent C.

psittaci strains include “pneumo-enteritis” with respiratory signs, mucopurulent nasal

discharge, diarrhea, polyuria and dullness (fluffed feathers and lethargy). Yellow-greenish

diarrhea is a common clinical sign. Occasionally unilateral or bilateral

conjunctivitis/keratoconjunctivitis, lameness and central nervous system disturbances may

be observed. The precipitation of disease and intensity of clinical signs and mortality also

depend upon avian species, chlamydial strains, environmental conditions and concurrent or

secondary infections (7, 8, 32, 188, 210).

The human infection by C. psittaci is called “psittacosis”, which was first named as

“psittakos” a Greek word meaning parrot (129). In 1879, Ritter observed this disease

among human first time due to exposure to tropical pet birds and named it

“pneumotyphous” (72, 158). Later world pandemic occurred in 1929 and 1930 due to

shipment of exotic birds from Argentina to Europe and North America and the causative

agent was first demonstrated in smears from infected birds and human patients (14, 34, 49,

CHAPTER-III

61

111). Since then various sporadic cases or large outbreaks have been reported in many

countries (155, 197, 224). According to the Office International des Epizooties (IOE) data

on zoonotic cases by avian chlamydiosis (http://www.oie.int/hs2/report.asp) from 2000 to

2004 period, 545, 391, 412, 524 and 410 cases were reported by 13, 13, 15, 12 and 13

member countries, respectively. But many more case may have gone undiagnosed owing

to confusing symptoms and difficult diagnosis.

Primarily the clinical signs of human psittacosis varies from mild flu like symptoms

to severe atypical pneumonia with non productive cough, fever, muscular and joint pain

and headache (115, 197, 213, 224) but the secondary complications like myocarditis (109),

hepatitis (172), encephalitis/meningitis and neurological disturbances (29, 36, 138, 168),

skin erythema (66, 180) or multiple organ failure (201) can increase fatalities. Some

suspected human-to-human transmission has also been reported (85). In old literature

psittacosis is referred to all human illness caused by all species, which have formerly been

grouped under Chlamydia psittaci group, but we considered reports those specifically

mentioned avian contact or otherwise doubtful histories.

For studying the pathogenesis of chlamydial diseases, animal experimentation models

are mostly used, as the genetic manipulation of chlamydiae is not possible at present time.

Studies on relative virulence of C. psittaci strains of avian origin are very few. In 1958,

Page first studied pathogenicity of C. psittaci strains from turkey, psittacine and pigeon in

mice (141) and in turkeys (142) and reported that some turkey strains are more pathogenic.

Subsequently, there have been many reports of experimental animal infection with

chlamydiae both in attempt to provide animal models of human disease and to establish

etiological relationships between infection and disease. However, the use of animal

infections primarily to differentiate between chlamydial strains is limited. The mouse

toxicity protection test first provided evidence of strain variations within C. trachomatis (2,

62

15). Rodolakis and colleagues differentiated invasive and non-invasive strains of C.

abortus by means of mice footpad inoculation experiments and found that invasive strain

infect placenta of pregnant mice (159) and sheep (161). These invasive C. abortus strains

formed a distinct genetic cluster, whereas non-invasive strains formed different clusters on

the basis of restriction endonuclease analysis of DNA (162) and RFLP patterns of ompA

gene (38).

Variation in the infectivity of C. psittaci strains to different avian and mammalian

hosts was observed in an earlier study (143). Different genetically and serologically variant

strains of C. psittaci have been identified in the newly classified Chlamydophila psittaci

group (43). We also divide all genetically diverse strains into 4 lineages and 8 genetic

clusters in CHAPTER II on the basis of the composition of ompA gene encoding the

MOMP. Depending upon MOMP specific panels of Mab and AluI RFLP pattern of ompA

gene, 8 serotypes and 7 genotypes are known (4, 56, 60, 174, 206, 207). The comparative

virulence and pathogenesis of different C. psittaci strains (new group) responsible for

avian chlamydiosis and human psittacosis has not been investigated in detail yet. The C.

psittaci strain of serovar D (turkey strains) and serovar A (parakeet strain) were observed

to be more invasive in experimentally infected turkeys than was serovar B (pigeon strains)

(212).

The murine infection models provide an important set of tools to identify the host

and organism factors responsible for disease pathogenesis. Different inbred and knockout

mice strains are used to study disease mechanisms of human (16, 21, 133, 223) and animal

(22, 124, 159) chlamydial infections. Therefore, in this study we compared the virulence of

C. psittaci strains predominantly detected form avian species and human psittacosis and

belonging to different genetic groups using murine model. Four C. psittaci strains were

intranasally inoculated in BALB/c mice and appearance of clinical signs, mortalities

63

(LD50) and clearance of infection was analyzed. Two genotypes found mostly associated

with human psittacosis were found to have low LD50 for BALB/c mice.


Chlamydiae: The 4 C. psittaci strains tested were, Nosé strain, isolated from a

budgerigar (Melopsittacus undulatus) that was in-contact with a human psittacosis patient

and was kindly provided by Prof. A. Matsumoto; MN/cal10 strain (49), isolated from

human/ferret in a human psittacosis outbreak; Mat116 strain isolated from a chestnut

fronted macaw (Ara severa) in an avian chlamydiosis out break in Japan (This study);

Borg strain (ATCC VR-601), isolated from the lung of a human psittacosis patient (140).

Chlamydial culture and purification of EBs: L-suspension cell line was used for

multiplying C. psittaci strains (50, 194). The cells were maintained in Eagle’s minimum

essential medium-1 (MEM-1) (Nissui, Japan) containing 5% fetal bovine serum (FBS) and

10 µg/ml of gentamicin. The EBs were harvested 3 to 4 days after inoculation and partially

purified as described in CHAPTER II by procedure reported by Tamura and Higashi (194).

The aliquots were stored at -80°C and later were used for determining stock EB

concentrations and for mice inoculations. The aliquots once thawed were not used again.

Quantification of EBs: The EB concentration in the stock was determined by

counting inclusion-forming units (IFU). Two-fold serially diluted, 200 µl suspension of

each strain was inoculated in duplicate on confluent McCoy cell monolayers grown (as

described in CHAPTER II) on 14 mm diameter glass round cover slips in 24 well plate

(Nunc, Roskilde, Denmark). After 24 hr of incubation at 37°C in 5% CO2, the coverslips

were fixed in chilled acetone for 10 min at 4°C and then stained with chlamydia group

specific fluorescein isothiocyanate (FITC)-labeled Mab (Denka Seiken Co. Ltd, Tokyo,

64

Japan). IFU were counted in duplicate slips by using fluorescent microscope (Olympus,

model BX50, Tokyo, Japan) using 200 times magnification and concentrations of EBs in

the stocks were calculated.

Mice: Specific pathogen free, female, 6 to 8 weeks old BALB/c mice were

obtained from Japan SLC Inc. (Shizuoka, Japan). The mice were kept in filtered top cages

in isolators under sterilized conditions. The mice were housed under standard

environmental conditions and received sterilized food and water ad libitum in accordance

with the guidelines for animal experiments at Gifu University.

Experimental design and inoculations: Total 96 mice were divided into 4 groups,

each consisting of 24 mice for each of 4 C. psittaci strains. With in each group, 4 mice

were inoculated with single dose and kept together in one cage. Mice were inoculated with

0.5 IFU, 5 IFU, 50 IFU, 5!102 IFU, 5!10

3 IFU and 5!10

4 IFU per gm body weight. Mice

were inoculated intranasally using 2 µl of inoculum per gram body weight with designated

concentration of EBs. A group of 6 mice, inoculated with same volume of MEM, was kept

as control. Mice were observed for 21 days post inoculation. The LD50 was calculated as

the method of Reed and Muench (154). Lung, liver, spleen and heart tissues were collected

from mice died before 21 days and those survived after 21 days post inoculation, in PBS,

pH 7.4 for DNA extraction and in sucrose-phosphate-glutamate (SPG) medium (218 mM

sucrose, 3.8 mM KH2PO4, 4.9 mM K2HPO4, 4.9 mM L-glutamic acid, pH 7.4) (17)

containing kanamycin 100 µg/ml, streptomycin 50 µg/ml, gentamicin 10 µg/ml and

vencomycin 100 µg/ml for determining chlamydial burden in lung, liver and spleen. The

blood samples were also collected from survived animals for serum extraction and

measuring IgG. The experiment was repeated twice with 5!102 and 5!10

3 IFU dose rates

with the same procedure.

Quantification of chlamydial burden: The 20% suspensions of lung, liver and

65

spleen tissues in SPG were centrifuged at 1,000 ! g for 10 minutes at 4°C. After

appropriate dilutions, 200 µl of supernatant was inoculated in duplicate on confluent

McCoy cell monolayer grown on glass coverslips in 24 well plate and incubated for 24 hr

at 37°C in 5% CO2. The coverslips were fixed in chilled acetone and IFU were counted by

the same procedure as mentioned in EB quantification protocol using chlamydia group

specific FITC-labeled Mab (Denka Seiken Co. Ltd, Tokyo, Japan).

DNA extraction and nested PCR: The DNA from lung, liver and spleen tissues of

mice was extracted by phenol-chloroform-isoamyl procedure (171) and nested PCR was

done as reported before (33) for detecting the chlamydial DNA to ascertain the clearance

of infection after 21 days post inoculation at different inoculation dose levels of all 4

strains.

Microimmunofluorescence test: The chlamydia specific IgG were detected by

microimmunofluorescence test as described before (156, 169) with some modifications.

Briefly McCoy cells were grown on Teflon coated 24 well glass plates (Cel-line; Erie

Scientific Co., Newfield, New Jersey, USA.) by adding 20 µl of suspension containing 105

cells/ml of MEM-1 with 5% FBS on each well and incubating for 24 hr at 37°C in 5% CO2

under moist conditions. Then after washing slides with MEM-1, 20 µl of suspension

containing 105 IFU of C. psittaci per ml of MEM-1 with 5% FBS and 1 µg/ml of

cycloheximide was added on each well of the glass slides and incubated under moist

conditions at 37°C in 5% CO2 for 24 hr. Then after removing medium, the slides were air

dried and fixed with acetone for 20 min. Then 20 µl of two fold serially diluted serum,

starting from 1:8, was placed on each well and incubated for 60 min at 37°C in moist

chamber. After washing thrice with chilled PBS, pH 7.4, the slides were incubated with

ICN/Cappel FITC-labeled sheep anti-mouse IgG (whole molecule) (ICN Biomedicals, inc.

Aurora, Ohiyo, USA) for 60 min. The slides were washed again thrice with chilled PBS,

66

pH 7.4 followed by washing with water and finally observed under fluorescent microscope

(Olympus, model BX50, Tokyo, Japan) at 200 time magnification after mounting in

SlowFade light antifade solution (Molecular Probes, Eugene, Oregon, USA).

Preinoculation and serum of negative control mice were used as negative control.

Statistical analysis: The analysis of body weight variation among different groups

of mice was done by comparing them with control group at different time points using two

tailed Student’s t-test and Mann-Whitney U-test. Results were considered significantly

different at the level of P value <0.05.

RESULTS

Different indices of virulence like the patterns of appearance/resolution of clinical

signs, body weight variations, mortalities and seroconversions at different doses of

inoculations with Nosé, MN, Mat116 and Borg strains of C. psittaci were monitored. The

data of repeated experiments was pooled in final analysis.

Clinical signs: The mice were observed for appearance of clinical signs like

general weakness, lethargy, ruffling of fur and respiratory signs. The scoring and pattern of

clinical sign are shown in Fig. 12. The appearance of clinical signs started on second to

third day post inoculation and the intensity was peaked at 4 to 5 days and persisted up to 9

to 10 days post inoculation among all 4 strains. At the dose rate of 5!103 and 5!10

4

IFU/gm body weight, all 4 strains showed the same intensity of clinical signs but at 50 and

5!102 IFU/gm body weight dose rate, MN showed milder clinical signs as compared to

Nosé, Mat116 and Borg strains. No clinical signs or very mild and transient signs of

disease were observed in all strains at dose of 0.5 and 5 IFU/gm body weight. The

resolution of clinical signs started after 12 days post inoculation and complete recovery

67

Fig. 12. The bar diagrams showing the patterns of appearance and resolution of clinical

signs in BALB/c mice inoculated intranasally with 0.5 IFU, 5 IFU, 50 IFU, 5!102 IFU,

5!103 IFU and 5!10

4 IFU per gram body weight with Nosé, MN, Mat116 and Borg

strains of C. psittaci. The 0-day indicates inoculation day and animals were observed for 21

days. The score 0: no clinical signs or normal, 1: mild weakness, lethargy and ruffling of

fur, 2: weakness, lethargy and ruffling of fur, 3: severe weakness, lethargy and ruffling of

fur along with labored breathing and hunched back, 4: death. The numbers of mice died

out of total inoculated are shown in the parentheses for each dose and strain. The control

group (inoculated with MEM-1) did not show any clinical signs. Each bar represents data

of a single mouse at particular time point.

0 3 6 9 12 15 18 21

Score

Score

0 3 6 9 12 15 18 21

Days

0 3 6 12 15 18 21

0 3 6 9 12 15 18

219

Score

0 3 6 12 15 18

21

9

0 3 6 12 15 189

0 3 6 12 15 18 219

0 3 6 12 15 18 219

Days

0 3 6 12 15 18 219

0 3 6 12 15 18 219

0 3 6 12 15 18 219

0 3 6 12 15 18 219

Days

Score

0 3 6 12 15 18 219

0 3 6 12 15 18 219

0

1

2

3

4

Sco

re

0 3 6 12 15 18 219

0

1

2

3

4

50 I

FU

Sco

re

0 3 6 12 15 18 219

Nosé MN Mat116 Borg

Days

Score

0

1

2

3

4

5 IF

U

Sco

re

0 3 6 12 15 18 219

0 3 6 12 15 18 219

0 3 6 12 15 18 219

0 3 6 12 15 18 219

0 3 6 12 15 18 219

0 3 6 12 15 18 219

0 3 6 12 15 18 219

0.5

IFU

Score

0

1

2

3

4

Sco

re

0 3 6 12 15 18 219

0

1

2

3

4

Sco

re

0

1

2

3

4

Sco

re

5×10

2 IF

U5×

103 I

FU

5×10

4 IF

U

21

(0/4) (0/4) (0/4) (0/4)

(0/4) (0/4) (0/4) (0/4)

(0/4) (0/4) (0/4) (0/4)

(0/8) (0/8) (2/8) (1/8)

(5/8) (2/8) (2/8) (3/8)

(4/4) (2/4) (2/4) (4/4)

69

Fig. 13. Mean relative body weight variations among the BALB/c mice inoculated with Nosé,

MN, Mat116 and Borg strains of C. psittaci. Mice were inoculated with 0.5 IFU (— line), 5

IFU (— line), 50 IFU (— line), 5!102 IFU (— line), 5!10

3 IFU (— line) and 5!10

4 IFU

(—line) per gram body weight on 0-day and observed for 21 days post infection. At each

time point, represented by 4 to 8 mice, P <0.05 was compared to control group (inoculated

with MEM-1 and shown by — line) using Student’s t-Test and Mann-Whitney U-Test. The

statistically significant time points are marked with asterisk (*). The error bar shows the

standard error. The points of mortality are shown by delta marks (") and its numbers

represent mice died at that point of time.

(A) Nosé strain

(B) MN/cal110 strain

(C) Mat116 strain

(D) Borg strain

72

was observed before 21 days, when experiment was terminated (Fig. 12).

Relative body weight variations: The relative body weight variations after the

inoculation with each strain is shown in Fig. 13. MN strain showed less variation in the

relative body weight at higher doses as compared to Nosé, Mat116 and Borg strains.

Mortality pattern and LD50: All the 4 strains were lethal to BALB/c mice at

various dose rates. No particular pattern of mortality was observed among all 4 strains. The

LD50 of Nosé, MN, Mat116 and Borg were observed to be 3.1!103, 19!10

3, 12!10

3 and

6.3!103 IFU/gm body weight, respectively.

Chlamydial burden among dead and recovered mice: To study the

pneumotropism and lethal lung burden, chlamydial EBs per gram of lung tissue of dead

mice were determined among mice inoculated with 5!103

IFU/gm body weight. At this

dose rate both mortalities and recoveries of mice were observed in all 4 strains. Among all

4 strains Borg strains multiplied in murine lung tissues rapidly and reached to the level of

35.7 ! 105 to 71.9 ! 10

5 IFU/gm body weight (Table 13). Among dead mice, infection also

spread to other primary organs like liver and spleen but the extent of multiplication was

found less than the primary target organ (lung).

Clearance of chlamydial infection: Samples from all dose level from all strains

were also screened by nested PCR to test the clearance of chlamydial infection from lung

liver and spleen after 21 days of infection. All samples except one infected with Nosé

strain, were found PCR negative (Table 13). This indicated that all the C. psittaci strains

irrespective of genetic group followed the same disease pattern in murine lung infection

model.

Chlamydia specific IgG immune response: The geometric mean titers of the

chlamydia specific IgG among recovered mice are shown in Table 14. The data showed

dose dependent increase in IgG tires among all 4 strains. It also showed that even lowest

73

Table 13. Chlamydial burden in lung, liver and spleen among the recovered and dead BALB/c

mice, inoculated intranasally with 5!103 IFU/gm body weight of Nosé, MN, Mat116 and Borg

strains of C. psittaci.

Strains Micea Chlamydia burden

b Anti-

chlamydial

Lung Liver Spleen IgG titers

IFU/gm PCRc IFU/gm PCR

c IFU/gm PCR

c

Nosé D5 <1 - - - - - 512

D6 <1 - - - - - 1!103

D18 1.2 ! 103 + <1 - <1 - 512

D7" 8.2 ! 105 + 8 !10

2 + 9.5 ! 10

2 + NT

D8" 10.5 ! 105

+ 6 ! 102 + 1.4 ! 10

3 + NT

D17" 6.5 ! 105

+ <1 + 2 ! 102 + NT

D19" 4.4 ! 105 + 1.2 ! 10

3 + 1.5 ! 10

2 + NT

D20" 6.4 ! 105 + 2.5 ! 10

2 + 1.5 ! 10

2 + NT

MN C5 <1 - <1 - <1 - 256

C6 <1 - <1 - <1 - 256

C8 <1 - <1 - <1 - 512

C17 <1 - <1 - <1 - 128

C19 <1 - <1 - <1 - 256

C20 <1 - <1 - <1 - 256

C7" 8 ! 105

+ 9.5 ! 102 + 1.1 ! 10

3 + NT

C18" 2.9 ! 105 + 5 ! 10

2 + 4 ! 10 + NT

Mat116 A7 <1 - <1 - <1 - 256

A8 <1 - <1 - <1 - 512

A17 <1 - <1 - <1 - 256

A18 <1 - <1 - <1 - 1!103

A19 <1 - <1 - <1 - 1!103

A20 <1 - <1 - <1 - 512

A5" 7.8 ! 105 + 8.5 ! 10

2 + <1 + NT

A6" 14.4 ! 105 + <1 + 2 ! 10

2 + NT

A14" 11.4 ! 105 + 1 ! 10

4 + 8.2 ! 10

3 + NT

Borg B6 <1 - - - - - 512

B8 <1 - - - - - 256

B17 <1 - <1 - <1 - 1!103

B18 <1 - <1 - <1 - 512

B20 <1 - <1 - <1 - 512

B5" 53.1 ! 105 + 1 ! 10

3 + 3.5 ! 10

3 + NT

B7" 71.9 ! 105 + 1.9 ! 10

3 + 4.7 ! 10

3 + NT

B19" 35.7 ! 105 + 7.2 ! 10

3 + 2 ! 10

3 + NT

a Dead mice are marked by asterisk.

b IFU count is the averages of duplicate samples and shown as numbers per gram of tissue and less

than one (<1) indicates no IFU in 200µl of 20% tissue suspension. c PCR positive samples are shown as plus sign (+) and PCR negative samples as negative signs (-).

Samples of all recovered mice, sacrificed after 21 days post infection, were found PCR negative for

all dose groups.

NT indicates not tested.

74

Table 14. Geometric mean of IgG titers detected among recovered BALB/c mice after intranasal

inoculation with C. psittaci strains. The numbers of tested sera (n) are given in parentheses.

C. psittaci Dose per gram body weight

a

strains

0.5 IFU 5 IFU 50 IFU 5!102 IFU 5!10

3 IFU 5!10

4 IFU

Nosé 65.5 (n=4) 138.8 (n=4) 372.2 (n=4) 421.0 (n=8) 608.9 (n=3) All mice died

MN 11.3 (n=4) 22.6 (n=4) 107.6 (n=4) 152.2 (n=8) 256.0 (n=6) 512.0 (n=2)

Mat116 22.6 (n=4) 26.9 (n=4) 53.8 (n=4) 143.7 (n=6) 512.0 (n=6) 724.1 (n=2)

Borg 9.5 (n=4) 26.9 (n=4) 53.8 (n=4) 312.1 (n=7) 512.0 (n=5) All mice died

a The dose rate is inclusion forming units (IFU) per gram body weight of mouse.

dose of 0.5 IFU/gm body weight can leads to seroconversion among inoculated mice.

DISCUSION

The financial losses resulting from the C. psittaci infections, particularly those in

the poultry and pet industries, combined with the fact that this is the most common animal

chlamydiosis transmissible to human beings, highlights the economic importance and

public health significance of the avian chlamydiosis (39, 115, 146, 181). Recent molecular

epidemiological studies have shown the genetic variations among C. psittaci strains (33,

51, 79, 191, 205). It was also observed that though many strains are prevalent among avian

fauna, only few types of strains are predominantly responsible for avian chlamydiosis

outbreaks and human psittacosis cases (33) (CHAPTER II). Therefore, to know the relative

virulence of the 4 representative strains of C. psittaci, we used BALB/c mouse model as it

would not affect the avian species specific pathogenicity of some strains and results may

be more representative of the virulence to non-avian hosts i.e. zoonotic infection of human

beings. Furthermore, in this study, intra nasal route of inoculation was used with varying

75

doses to simulate the natural route of infection (mostly infection is by inhalations of

infectious aerosol) (142) and extent of exposure which represent the amount of infectious

material inhaled that is related to the final outcome human psittacosis infection (140, 197,

224).

It was observed that the Nosé strain that represent a group of strains, mostly

prevalent among psittacine birds, is most lethal to the BALB/c mice. The various reports in

the literature concerning human psittacosis and horizontal transmission of infection to in-

contact animals, support our findings that similar or closely related types of strains are

highly virulent and transmissible to non-avian hosts (37, 68, 107, 116, 197, 209, 211, 224).

The second most lethal strain to mice was Borg, which represents C. psittaci strains mostly

prevalent among poultry birds. This type of strains may be associated with human

psittacosis cases in the poultry-industry workers (8, 117, 127, 146). Two other strains,

Mat116 and MN/cal10 were also found to be lethal to BALB/c mice only at higher dose

rate, suggesting that such strains can be potentially pathogenic to avian and non avian hosts

when the exposure to the source of infection is prolonged.

The monitoring of appearance and resolution of clinical signs during the course of

disease, at varying doses of inoculation, also indicated that Nosé and Borg strains produce

severe clinical disease in mice even at lower dose rate as compared to MN. Therefore, the

C. psittaci strains of these genetic groups may induce clinical disease among in-contact

human or other mammalian hosts even after brief exposure to the source of infection.

However, similarity in the pattern of appearance/resolution of clinical signs in all tested 4

strains indicated that the disease pathogenesis and protective immune response mounted by

hosts to all types of C. psittaci strains in murine lung infection model may be the same.

This was further confirmed by testing IgG immune response and chlamydial burden in

lung, liver and spleen among recovered and died mice. It showed that IgG titers are dose

76

dependent and chlamydial infection is almost cured within 21 days. The IgG also appear at

10 to 14 days of infection in mice, experimentally infected with human biovars of C.

trachomatis and C. pneumoniae (73, 104, 223). The variations in levels of morbidity and

mortality indicate that, though the pattern of resolution of infection is the same in all C.

psittaci strains, the level of protection to primary infection is variable. It may be due to the

difference in virulence factors associated with infecting strain.

Human psittacosis is not a major public health problem in term of numbers of cases

reported but it remains an important occupational hazard to the workers of poultry

industries, pet shops or aviaries staff, veterinarians and avian pets owners (39, 115, 146).

Many cases of human psittacosis occur without any history of avian contacts. Therefore, it

is necessary to test for C. psittaci in suspected cases of psittacosis and atypical pneumonia

(39, 224). Our study will provide a platform for further studies to identify the virulent

factors associated with highly pathogenic strains for better protection of avian and human

health.

SUMMARY

For evaluating the degree of virulence of C. psittaci strains predominantly

responsible for avian chlamydiosis and human psittacosis, BALB/c mice were used as

experimental infection model. Four representative C. psittaci strains: Nosé, MN, Mat116

and Borg were intranasally inoculated at dose rate of 0.5 IFU, 5 IFU, 50 IFU, 5!102 IFU,

5!103 IFU and 5!10

4 IFU per gram body weight and different parameters of virulence

were monitored. The LD50 of Nosé, MN, Mat116 and Borg strains were 3.1!103, 19!10

3,

12!103 and 6.3!10

3 IFU/gm body weight, respectively. The intensity of clinical signs

against the infectious doses of 5!103 and 5!10

4 IFU/gm body weight was the same among

77

the 4 strains, however, at 50 and 5!102 IFU/gm body weight doses, the MN strain showed

milder clinical signs. The animals inoculated with the MN strain at higher doses also

showed less variations in the relative body weights as compared to those inoculated with

Nosé, Mat116 and Borg strains. Among the 4 strains, Borg strain rapidly multiplied in

murine lung tissues and infection reached up to 35.7 to 71.9 ! 105 IFU/gm body weight

level at the point of death. Lateral spread of infection to other primary organs including

liver and spleen was also observed. The complete recovery and clearance of chlamydial

infection was verified at 21 days post infection by detecting chlamydial DNA and/or

chlamydial IFU in lung, liver and spleen tissues. Chlamydia specific IgG immune response

was found to be dose dependent. This study indicated that the C. psittaci strains frequently

associated with avian and human psittacosis cases were highly virulent to mice and that the

immune responses lead to variable degrees of protection. Our murine lung infection model

can be used for further virulence analysis studies at molecular level.

78

PART II

Investigation of the Emergence of Chlamydial Infection Among Animals and

Human Disease Conditions

79

Involvement of multiple Chlamydia suis genotypes in porcine conjunctivitis

in commercial farms of Japan

INTRODUCTION

Porcine chlamydial infections are either clinical or subclinical in nature. In the past,

Chlamydia trachomatis like strains were detected from various types of porcine specimens

in USA (99, 165, 183). These genetically distinct and host specific strains have been

classified as Chlamydia suis under family Chlamydiaceae in recent classification on the

basis of 16S rRNA gene (43). C. suis has been mainly detected either from conjunctivitis,

pneumonitis, pericarditis, vaginitis, mastitis and enteritis cases or from lymph nodes,

semen, feces and lungs of normal pigs (82, 84, 99, 166, 196). Other chlamydiae such as

Chlamydophila abortus, Chlamydophila pecorum, Chlamydophila psittaci and Chlamydia-

like organisms are also reported in pigs with respiratory and reproductive tract disorders,

polyarthritis and semen from healthy boars (82, 84, 178, 179, 196). Mixed infection of C.

suis with C. pecorum in aborted porcine fetal livers, and with C. abortus in intestinal,

respiratory and reproductive tract samples have also been reported (82, 179).

Epidemiological studies on porcine chlamydiosis have indicated high prevalence

rate among sick and normal pigs in Germany (82, 84), USA (95, 165) and Switzerland

(178, 179). Recent studies have also indicated prevalence of chlamydial infection among

both male and female porcine used for breeding purpose in Belgium, Germany and

Switzerland (100, 196, 211). In Japan there is only one report of abortion due to

Chlamydia psittaci in a pig farm (139) and another report of prevalence of complement

CHAPTER-IV

80

fixing antibodies among 0.7% pig samples (58). There is no study that has identified

involvement of specific chlamydial species or strain in the porcine clinical infections.

For detection and identification of chlamydial species, 16S-23S ribosomal RNA

intergenic spacer region was reported as a suitable target (41, 44). The ompA gene that

encodes MOMP is also the most preferred target for chlamydial epidemiological studies

and analysis of genetic diversity of the chlamydiae detected directly from field samples

(33, 82, 96). Chlamydial ompA gene consists of genetically conserved domains (CD)

flanking 4 genetically variable domains (VD). The VDs of ompA gene are reported to have

species/strain specific sequences, which encode immunologically specific epitopes on

MOMP (12, 98). Genetic analysis of ompA gene of C. suis strains of lungs and intestinal

origin has shown 20% nucleotide variation (82).

In the present study, the etiology of frequent conjunctivitis incidences and some

reproductive disorder cases occurring among 5 commercial farms in 3 prefectures of Japan

was investigated. Initial screening was done by chlamydia group specific direct fluorescent

antibody test (FAT) on impression smears. Then followed by nested PCR tests targeting

16S-23S rRNA intergenic spacer region and ompA gene for detection, identification and

analysis of genetic diversity in chlamydial spp./strains. Complement fixation test (CFT)

was done to screen serum samples. Diverse C. suis genotypes were identified among

conjunctivitis cases by nucleotide sequences analysis of 16S-23S rRNA intergenic spacer

and ompA gene fragments.


Samples analyzed: A total of 61 samples were taken from 49 animals of 5

commercial pig farms in 3 prefectures of Japan from 2003 to 2005. The samples were 35

81

ophthalmic swabs taken from pigs with mucopurulent conjunctivitis and

keratoconjunctivitis (Fig. 14), 9 vaginal swabs from aborted animals, 2 tonsilar swabs and

3 seminal fluid samples from breeding boars. Twelve serum samples were also collected

(Table 15).

Table 15. List of the farms and samples analyzed.

Farms (prefectures) Disease condition Type of Sample

samples no.

Farm-I (Chiba) Conjunctivitis Eye swabs 10

Normal Tonsillar swabs 2

Farm-II (Miyagi) Conjunctivitis Eye swabs 10

Abortion Vaginal swabs 9

Normal Seminal fluid 3

Abortion and normal Serum samples 12

Farm-III (Kanagawa) Conjunctivitis Eye swabs 5

Farm-IV (Kanagawa) Conjunctivitis Eye swabs 5

Farm-V (Kanagawa) Conjunctivitis Eye swabs 5

Total 61

Chlamydial and other bacterial species: For standardizing the PCR test, DNA

was extracted from S45 strain of C. suis (ATCC VR 1474) and other Chlamydophila spp.

viz. C. psittaci- 6BC and MN strains, C. felis- FePn Baker strain, C. abortus- B577 strain

and C. caviae- GPIC strain. DNA of Escherichia coli, Staphylococcus aureus,

Pseudomonas sp., and Proteus sp. was also extracted and used for testing specificity of the

PCR. The chlamydial and bacterial strains were obtained from the Laboratory of

Veterinary Microbiology, Gifu University, Japan.

Complement fixation test (CFT): Total 12 serum samples collected from a pig

farm (farm-II, Table 15) were tested by CFT using Chlamydia psittaci CFT kit (Denka

Seiken Co. Ltd, Tokyo, Japan) according to the instructions of the manufacturer.

82

Fig. 14. A domesticated pig (Farm-I, Sample ID PG18) with keratoconjunctivitis and

mucopurulent ocular discharge tested C. suis. positive by nested PCR.

Direct fluorescent antibody test (FAT): Total 9 ophthalmic swabs and 9 vaginal

swabs were stained by chlamydia group specific FITC-coupled Mab (Denka Seiken Co.

Ltd, Tokyo, Japan) according to the manufacture’s protocol and chlamydial elementary

bodies (EBs)/inclusions were observed by fluorescent microscope (Olympus, model BX50,

Tokyo, Japan) using 200 times magnification.

DNA extraction: DNA was extracted using SepaGene DNA extraction kit (Sanko

Junyaku, Tokyo, Japan) following manufacture’s instructions. Briefly ophthalmic, vaginal

and tonsillar swabs or 200 µl of seminal fluid were resuspended in 700 µl of phosphate-

buffered saline (PBS) pH 7.4 and centrifuged at 16,000 ! g for 30 min at 4°C and

sediments were processed further for DNA extraction. DNA pallets were finally

83

resuspended in 30 µl of Tris-EDTA (TE) pH 7.4 (100 mM Tris-HCl, pH 7.4 and 10 mM

EDTA, pH 8.0) and stored at -30°C until use.

Nested PCR tests: The PCR test targeting 16S-23S rRNA intergenic spacer region

was done as reported before (41, 44) with some modifications. 16SF2 and 23R primers

were used as outer pair and 16SF3 and IGR as inner pair to amplify 16S-23S rRNA

intergenic spacer segment. The Chlamydophila genus specific test was also performed as

reported before (33). Chlamydia genus specific nested PCR was standardized in this study.

The oligonucleotide primer sequences used in the study are shown in Table 16. PCR

primers were synthesized by Rikaken Co., Nagoya, Japan. The conditions for maximum

DNA amplification were adjusted using variable annealing temperatures, primer

concentrations and thermocycling conditions. All the PCR tests were performed in 50 µl

reaction mixture containing 0.20 µM of each forward and reverse primers, 250 µM of each

dATP, dTTP, dGTP, dCTP, 100 µM of Mg2+

in buffer and 2.5 units of TaKaRa Ex-Taq

(Takara Bio Inc., Shiga, Japan) and 2.0 to 5.0 µg of template DNA of each sample. DNA

of C. suis S45 strain was used as a positive control. Five microliters of the first step PCR

product was used for the second step PCR. The thermocycling conditions used in both

steps of Chlamydia genus specific PCR were: 3 min at 94°C then 35 cycles of 30 sec at

94°C, 20 sec annealing at 58°C and extension for 90 sec at 72°C (60 sec in second step

PCR), followed by 5 min at 72°C and soaking at 4°C. For the amplification of 16S-23S

rRNA intergenic spacer region, same thermocyclic conditions were used except annealing

for 30 sec at 55°C (50°C in second step PCR) and extension of 60 sec at 72°C (30 sec in

second step PCR) during 35 cycles. PBS and water were used as negative controls.

Sensitivity and specificity were verified under the determined conditions with DNA

templates of different bacteria and Chlamydophila spp.

Table 16. Oligonucleotide primers used in the study.

Primer name Nucleotide sequence Positionsa

Melting Reference

temperature (°C)

16S-23S rRNA intergenic spacer regionb:

16SF2 5'-CCGCCCGTCACATCATGG-3' 1398-1415 (16S rRNA) 60-62 (41)

23R 5'-TACTAAGATGTTTCAGTTC-3' 212-193 (23S rRNA) 47-49

16SF3 5'-TCGTAACAAGGTAGCCC-3' 1496-1512 (16Sr RNA) 52-54

IGR 5'-AGCTCTTA(T/G/A)(C/T)AACTTGGTCTGTA-3' 23-1 (23S rRNA) 53-55 (44)

ompA gene:

Genus Chlamydia specific This study

CS-1PF 5'-GGAATCCTGCTGAACCAAGCCTTATGAT-3' 77-104 61-63

CS-1PR 5'-GCGAGTCTCAACAGTAACTGCGTATT-3' 1107-1082 60-62

CS-2PF 5'-GGCGTTGAATATTTGGGATCGTTTTG-3' 366-391 58-60

CS-2PR 5'-CTTGCTCGAGACCATTTAACTCCAATG-3' 872-846 59-61

Genus Chlamydophila specific (33)

CMGP-1F 5'-CCTTGTGATCCTTGCGCTACTTG-3' 138-159 60-62

CMGP-1R 5'-GTGAGCAGCTCTTTCGTTGAT-3' 1184-1164 57-60

CMGP-2F 5'-GCCTTAAACATCTGGGATCG-3' 384-403 56

CMPG-2R 5'-GCACAACCACATTCCCATAAAG-3' 634-613 57

a The positions of the primers are based on the ompA gene sequence of Chlamydia suis, PCLH197 strain (accession no. AJ440241) and

Chlamydophila psittaci, 6BC strain (accession no. X56980).b The primer positions are based on 16S rRNA and 23S rRNA genes of R22 strain of C. suis (accession no. U68420).

85

Cloning of PCR product and sequencing: The second step PCR products were

purified by gel electrophoresis using low melting agarose gel in Tris-acetate-EDTA (TAE)

buffer, pH 7.4 followed by QIAquick Gel Extraction kit (Qiagen, Hilden, Germany). The

DNA fragments were cloned in pGEM-T vector (Promega, Madison, Wisconsin) and

DH5! strain of E. coli (Tyobo Co., Osaka, Japan) was transformed by heat shock method

(171). From each PCR product, 5 clones with expected size DNA insert were taken for

sequencing. The sequencing was done using the dye-terminator method and performed by

a commercial resource (Dragon Genomics Co., Mie, Japan). Both strands were read. The

sequences were assembled and edited using Genetyx-Mac/ATSQ 4.2.3 and Genetyx-Mac,

version 13.0.6 (SDC, Tokyo, Japan).

Analysis of sequences and construction of phylogenetic trees: The chlamydial

species and strains were identified by NCBI-BLAST (http://www.ncbi.nlm.nih.gov) search

of nucleotide sequences of 16S-23S rRNA spacer region and ompA gene. For phylogenetic

analysis, the 16S-23S rRNA spacer region and ompA gene sequences of C. suis strains and

each representative species of genus Chlamydia were retrieved from the DDBJ. Multiple

alignments of the trimmed sequences were done using ClustalX, version 1.83 (198).

Phylogenetic analysis was done with programs in the PHYLIP (version. 3.6a3;

[http://evolution.genetics.washington.edu/phylip.html]). The distance matrix between

species was computed by DNADIST and clustering of lineages was done by NEIGHBOR

using neighbor-joining method. The bootstrap values were calculated to evaluate the

branching reliability of trees from a consensus tree constructed by generating 1,000

random data sets using SEQBOOT.

86

RESULTS

Epidemiological studies: In the preliminary screening, impression smears from

conjunctival swabs (n=9) and vaginal swabs (n=9) from animals with severe clinical signs

were examined with FAT. Chlamydial EBs and/or inclusions were detected from 33.3% (3

out of 9) conjunctivitis samples only. Then all the samples (n=49) were examined by using

nested PCR tests. By 16S-23S rRNA intergenic spacer and Chlamydia genus specific PCR,

22 out of 49 (44.9%) samples were found positive. All PCR positive samples were accrued

from conjunctivitis cases. In the conjunctivitis cases, 22 out of 35 (62.9%) ophthalmic

swab samples were positive including all those which were FAT positive. All samples

were found negative by Chlamydophila genus specific nested PCR. No chlamydial

infection was detected among samples from tonsillar swabs (Table 17).

Table 17. The 16S-23S rRNA intergenic spacer and ompA gene based nested PCR results for detection of

Chlamydia species and Chlamydophila species.

Farms Type of

Total

samples PCR positive sample

Chlamydial

species

samples tested Intergenic Chlamydia Chlamydophila identified

spacer

primers

specific

primers

specific

primers

Farm-I Eye swabs 10 6 (60%) 6 (60%) 0 C. suis

Tonsillar

swabs 2 0 0 0

Farm-II Eye swabs 10 3 (30%) 3 (30%) 0 C. suis

Vaginal swabs 9 0 0 0

Seminal fluid 3 0 0 0

Farm-III Eye swabs 5 4 (80%) 4 (80%) 0 C. suis

Farm-IV Eye swabs 5 5 (100%) 5 (100%) 0 C. suis

Farm-V Eye swabs 5 4 (80%) 4 (80%) 0 C. suis

Total 49 22 (44.9%) 22 (44.9%)

87

The vaginal swab samples from 9 aborted sows and seminal fluid from 3 breeding

boars were found negative by all PCR tests. The serum samples analysis of these 12

animals (aborted sows=9 and normal breeding boars=3) by CFT showed 5 aborted sows

with antichlamydial immmunoglobulins titers of more than 1:8 (1:8 for 2 animals and 1:16

for 3 animals) indicating possible non-chlamydial etiology of current reproductive

problems but may have previously exposed to the chlamydial infection.

Genetic variation analysis: The analysis of 238 to 241 bp nucleotide sequence of

16S-23S rRNA intergenic spacer region of all PCR positive samples by NCBI-BLAST

(http://www.ncbi.nlm.nih.gov) search and phylogenetic clustering in NJ tree confirmed the

involvement of C. suis (Fig. 15 and Table 17). Total 9 variants of 16S-23S rRNA

intergenic spacer region nucleotide sequences with less than 3% differences were found

(Fig. 16) and were designated as sequence types (Table 18-a). To further confirm the

genetic diversity among C. suis strain involved in clinical cases of conjunctivitis,

nucleotide sequence analysis of ompA gene fragment of 454 to 463 bp that includes VD2

and VD3 regions was done. Two PCR positive samples from each farm, including those

with multiple 16S-23S rRNA intergenic spacer region nucleotide sequences, were

randomly selected and sequenced. Twenty types of nucleotide sequences (sequence types)

were detected (Table 18-b). All the C. suis strains showed up to 22% nucleotide variations

in the sequenced region.

The predicted amino acid sequence alignment of all the detected and already

known C. suis strains in the sequenced fragment of ompA gene (151 to 154 amino acids)

revealed the high genetic variations in the form of amino acid substitutions, insertions and

deletions leading to gaps formation (varies from 0 to 3 in numbers) in VD2 along with

some conserved motifs of amino acid residues in VD2 and VD3 region (Fig. 17).

On the basis of the sequence variation in 16S-23S rRNA intergenic spacer region,

Chlamydia muridarum-SFPD (U68437)

Chlamydia trachomatis-A/HAR-13 (CP000051)

Chlamydia trachomatis-B/TW-5/OT (U68440)

Chlamydia trachomatis-D/UW-3/CX (AE001273)

Chlamydia trachomatis-F/IC/CAL3 (U68442)

Chlamydia trachomatis- L2/434/BU (U68443)

Chlamydophila psittaci-MN (U68453)

CSR0503CSR0515

S45 (U73110)

CSR0304CSR0305

CSR0301

CSR0517, R19 (AF481047), R22 (U68420)

R24 (U68428)R27 (U68429)

H5 (U68427)

CSR0401CSR0402

CSR0505

MS04 (DQ118376)

CSR0504

CSR0511 CSR0502CSR0501CSR0507

Chlamydia muridarum-Nigg (AE002160)

0.005 units

Chlamydia suis

Fig. 15. The NJ tree of 16S-23S rRNA intergenic spacer region of detected (in bold letters) and knownstrains of C. suis showing separate grouping in relation to the C. trachomatis and C. muridarum species. The branch of C. psittaci is reduced three times. The accession numbers of the detected C. suis strains are: CSR0301-AB283016, CSR0303-AB283017, CSR0304-AB283018, CSR0305-AB283019, CSR0401-AB283006, CSR0402-AB284054, CSR0501-AB283007, CSR0502-AB283008, CSR0503-AB283009, CSR0504-AB283010, CSR0505-AB283011, CSR0507-AB283012, CSR0511-AB283013, CSR0515-AB283014, CSR0518-AB283015 and those of known species and strains are shown against each strain in parentheses in figure. The bootstrap values are shown against each node. The relative genetic distance isshown in 0.005 unit bar.

CSR030393%

54.4%99.8%

99.8%

75%

61.8%

90

Table 18. Distribution of C. suis sequence types in different farms and animals based on (a) 16S-23S rRNA

intergenic spacer region and (b) ompA gene. The ompA gene based genotypic clusters depending on gaps in

the VD2 region are also shown.

(a) 6S-23S rRNA intergenic spacer region

Farms (prefectures) Sample IDa Sequence

b Strains Accession no.

types

Farm-I (Chiba) PG18 1 CSR0301 AB283016

PG21 1 CSR0303 AB283017

2 CSR0304 AB283018

3 CSR0305 AB283019

Farm-II (Miyagi) PG1 4 CSR0401 AB283006

PG2 4 CSR0402 AB284054

Farm-III (Kanagawa) PG5 4 CSR0501 AB283007

5 CSR0502 AB283008

6 CSR0503 AB283009

7 CSR0504 AB283010

PG6 4 CSR0505 AB283011

Farm-IV (Kanagawa) PG7 4 CSR0507 AB283012

PG8 4 CSR0511 AB283013

Farm-V (Kanagawa) PG9 8 CSR0515 AB283014

PG10 9 CSR0518 AB283015

(b) ompA gene

Farms (prefectures) Sample IDa Sequence

b Genetic Strains Accession no.

types clusterc

Farm-I (Chiba) PG18 1 A CS0301 AB270719

2 A CS0302 AB270720

PG21 1 A CS0303 AB270721

2 A CS0304 AB270722

Farm-II (Miyagi) PG1 3 B CS0401 AB270723

PG2 3 B CS0402 AB270724

Farm-III (Kanagawa) PG5 4 A CS0501 AB270725

5 A CS0502 AB270726

6 D CS0503 AB270727

PG6 7 A CS0504 AB270728

8 A CS0505 AB270729

9 D CS0506 AB270730

Farm-IV (Kanagawa) PG7 10 A CS0507 AB270731

3 B CS0508 AB270732

11 A CS0509 AB270733

1 A CS0510 AB270734

PG8 12 D CS0511 AB270735

13 D CS0512 AB270736

14 D CS0513 AB270737

15 D CS0514 AB270738

Farm-V (Kanagawa) PG9 16 B CS0515 AB270739

17 B CS0516 AB270740

18 A CS0517 AB270741

PG10 19 D CS0518 AB270742

20 D CS0519 AB270743

a Represents the independent sample taken from the individual animal.

b The sequences with same numerical numbers are having 100% homologous nucleotide sequences. The

nucleotide sequence types with similar deduced amino acid sequences are bracketed together. c As grouped in Figs. 17 and 18.

91

only 2 animals (PG21 and PG5) were found harboring multiple genotypes of C. suis (Table

18-a), but ompA gene based screening detected much more cases of multiple genotypes

infection in individual animal or farm. The distribution of different ompA gene based C.

suis genotypes detected among 5 farms and 10 animals are shown in Table 18-b, Fig. 17

and Fig. 18. One animal of farm-I and farm-III each was found to have 3 and 4 types of C.

suis. In 4 farms, each animal was found to harbor 2 to 4 types of genetically varied C. suis

strains. Whereas, only one genotype was detected among animals of farm-II. The

genetically closely related sequence types 1 and 2 were detected from 2 animals of farm-I

and one animal (PG7) of farm-IV. However, animals of farm-III and farm-V and PG7

animal of farm-IV were found harboring highly diverse genotypes. These results indicate

the high genetic variation among the C. suis stains infecting either a single animal or

prevalent in the same farm. The overall numbers of genotypes detected based on sequence

analysis of 16S-23S rRNA intergenic spacer and ompA gene are shown in Table 19.

Table 19. Number of sequence types detected in different farms and animals based on the

16S-23S rRNA intergenic spacer region and ompA gene.

Farms (prefectures) Sample IDa Sequence types

b

Intergenic spacer ompA gene

Farm-I (Chiba) PG18 1 2

PG21 3 2

Farm-II (Miyagi) PG1 1 1

PG2 1 1

Farm-III (Kanagawa) PG5 4 3

PG6 1 3

Farm-IV (Kanagawa) PG7 1 4

PG8 1 4

Farm-V (Kanagawa) PG9 1 3

PG10 1 2

a Represents the independent sample taken from the individual animal.

b The sequence types with the same numerical numbers are having 100% homologous

nucleotide sequences.

92

Phylogenic analysis: All the detected and known strains of C. suis are clustered

together in a group vis-à-vis other Chlamydia spp. and representative Chlamydophila

psittaci strain in 16S-23S rRNA intergenic spacer based NJ tree (Fig. 15). For further

determination of epizootiologically predominant genotypes of C. suis, all the 25 detected

ompA gene sequences (20 variant types) were compared with all the known strains of C.

suis derived from lungs, enteric, nasal and ophthalmic samples either from healthy or

diseased animals. All the strains were broadly grouped into 4 clusters; named A, B, C and

D depending upon the numbers of gaps in the VD2 (Fig. 17 and 18). Genetic cluster A

strains have no gap whereas strains of clusters B, C and D have one, two and three gaps,

respectively in the VD2. Epizootiologically predominant clusters are D, A, C and B in

descending order. In this study, C. suis strains only belonging to genetic clusters A, B and

D could be detected. Distribution of these genetic clusters among 5 farms and screened

animals is shown in Table 18-b. The distributions of C. suis strains into 4 genetic clusters

do not related to host disease condition or tissue tropism.

DISCUSSION

C. suis is a recently identified pathogen of both domesticated and wild porcine (43,

84, 95, 165). C. suis has been demonstrated experimentally to be capable of causing acute

pneumonia (166, 167), conjunctivitis (163) and enteric lesions (164) in gnotobiotic pigs.

Limited epizootiological studies have indicated that C. suis infections in pigs are wide

spread but under diagnosed. For detection of porcine chlamydiosis particularly due to C.

suis, a sensitive ompA gene based PCR test was developed and used along with previously

reported broad range 16S-23S rRNA based PCR. In the present investigation, C. suis was

detected among 62.9% conjunctivitis samples by PCR, suggesting higher prevalence of C.

93

Fig. 17. Genetic clusters of C. suis strains detected in this study and reported in data bank

(referred by accession no.) based on the alignment of amino acid sequences in VD2 and

VD3 regions of ompA gene and variation in length of sequenced portion of ompA gene

owing to gaps in VD2 region. The corresponding nucleotide sequence types (from 1 to 20),

sample identification and farm numbers are also shown for each detected strain. The

portion within the boxes represents the genetically variable domains flanked by the

constant regions of ompA gene. Dots indicate the identical amino acids and hyphens

represent the gaps. Asterisks in the bottom line mark the conserved amino acid residues.

The alignment was done by ClustalX ver. 1.83

Consensus seq. VFCTLGATNGYLKGNSAAFNLVGLFGDT--ATSVAA-TDLPNVSLSQAVVELYTDTAFAWSVGARAALWECGCATLGASF

CS0301 1 ...........................DTQ..QM.QSKN..........I.............................. 80 1 I PG18CS0302 1 ...........................DTQ..QM.QSKN..........I.............................. 80 2 I PG18CS0303 1 ...........................DTQ..QM.QSKN..........I.............................. 80 1 I PG21CS0304 1 ...........................DTQ..QM.QSKN..........I.............................. 80 2 I PG21CS0502 1 .................S.........PTQINN..TAKT..........I.............................. 80 5 III PG5CS0504 1 ...........P.....S.........PTQ.AQ..TTKN..........I.............................. 80 7 III PG6CS0505 1 .................S.........PTQ.AQ..TTKN..........I.............................. 80 8 III PG6CS0507 1 ..............S..S........TKTQ.AQFNTAKIV........................................ 80 10 IV PG7CS0509 1 ...........................DTQ..QM.QSKN..........I..C.....T..................... 80 11 IV PG7CS0510 1 ...........................DTQ..QM.QSKN..........I.............................. 80 1 IV PG7CS0517 1 ...........................PSQLNN..TAKA..S...................................... 80 18 V PG9R27 1 .................S.........PTTVND..TAKT..........I..............K............... 80 Ref. ( accession no. AF269273)Roger130 1 .................S.........PTQVNN..SAKTI.........I.............................. 80 Ref. ( accession no. AF269277)MS04 1 ...........................PTQSSQ..TSKN..........I.............................. 80 Ref. ( accession no. DQ118378)pm39 1 ...........P.....S........TKTQ.SAFD.AKLV........D............................... 80 Ref. ( accession no. AJ004876)Roger132 1 .................S........TKTQ.SAFD.AKLV........................................ 80 Ref. ( accession no. AF269278)

CS0401 1 .................S.........NVD..T.TK-.SI.......S.I.............................. 79 3 II PG1CS0402 1 .................S.........NVD..T.TK-.SI.......S.I.............................. 79 3 II PG2CS0508 1 .................S.........NVD..T.TK-.SI.......S.I.............................. 79 3 IV PG7CS0515 1 ...........................DEN.AQPIG-.S...FA.D.S.I.............................. 79 16 V PG9CS0516 1 ...........................DEN.AQPIG-.S...FA.D.S.I.............................. 79 17 V PG9H7 1 .................S.........NANQST..Q-GSI.......S.I.............................. 79 Ref. ( accession no. AF269276)

R19 1 .................S........--ANE.TA.QA.AV.......S................................ 78 Ref. ( accession no. AF269270) H5 1 .................S........--ANE.TA.QA.AV.......S................................ 78 Ref. ( accession no. AF269275)S45 1 .................S........--ANE.TA.QA.AV.......S................................ 78 Ref. ( accession no. AF269274)32XII 1 .................S........--ANEQNA.GA.AV.....A.S................................ 78 Ref. ( accession no. AY687634)2V 1 ..........................--ATEQTAVLK.LV.......S................................ 78 Ref. ( accession no. AY687633)13VII 1 ..........................--ANDQNTLPE.AV..A..A.S................................ 78 Ref. ( accession no. AY687632)R22 1 ..........................--TNELTAQLN.LV.....A.S................................ 78 Ref. ( accession no. AF269271)

CS0501 1 ..........................L.--T...G.-........N........E......................... 77 4 III PG5CS0503 1 ..........................L.--T...G.-........N........E......................... 77 6 III PG5CS0506 1 ..........................L.--G.....-......N.T.................................. 77 9 III PG6CS0511 1 ..........................LN--VAT.GK-........N.................................. 77 12 IV PG8CS0512 1 ..........................LN--VAT.GK-........N.................................. 77 13 IV PG8CS0513 1 ..........................LN--VAT.GK-........N.................................. 77 14 IV PG8CS0514 1 .......A..................LN--VAT.GK-........N.................................. 77 15 IV PG8CS0518 1 ............R.............L.--T..A..-Q.......A........E......................... 77 19 V PG10CS0519 1 ..........................L.--T.....-Q.......T........E......................... 77 20 V PG10pm220 1 ..........................L.--T...G.-........N........E......................... 77 Ref. ( accession no. AJ005616)pmsh47 1 ..........................L.--T...G.-........N........E......................... 77 Ref. ( accession no. AJ004878)pm197 1 ..........................L.--T...G.-........N........E......................... 77 Ref. ( accession no. AJ004877)PCLH296 1 ..........................L.--T...G.-........N........E......................... 77 Ref. ( accession no. AJ004880)PCLH197 1 ..........................L.--T...G.-........N........E......................... 77 Ref. ( accession no. AJ440241)pmd1340 1 ..............D...........L.--T...G.-........N........E......................... 77 Ref. ( accession no. AJ004879)R24 1 ..........................L.--T.....-Q.......T........E......................... 77 Ref. ( accession no. AF269272)14V 1 ..........................L.--G.....-......N.T.................................. 77 Ref. ( accession no. AY687630)14VII 1 ..........................L.--G.....-......N.T.................................. 77 Ref. ( accession no. AY687631) *******.***..*.**.******** . .. . .*...*.*...**.*.***.*****.***************

Consensus seq. QYAQSKPKVEELNVLCNAAEFTINKPQGYVGQEFPLPETAGTSAATGTKDASIDYHEWQASLALSYRLNMFTP

CS0301 80 ...............................A....DR....D.............................. 153 1 I PG18CS0302 80 ...............................A....DR....D.............................. 153 2 I PG18CS0303 80 ...............................A....DR....D.............................. 153 1 I PG21CS0304 80 ...............................A....DR....D.............................. 153 2 I PG21CS0502 80 ...............................AD...ELK...D.............................. 153 5 III PG5CS0504 80 ..........................K.....A...EF....D.............................. 153 7 III PG6CS0505 80 ..........................K.....A...EF....D.............................. 153 8 III PG6CS0507 80 ..........................K...D.A...DI....E.............................. 153 10 IV PG7CS0509 80 ....................S..........A....DR....D.............................. 153 11 IV PG7CS0510 80 ...............................A....DR....D.............................. 153 1 IV PG7CS0517 80 ...............................A....ELK...D.............................. 153 18 V PG9R27 80 ..........................K...D.A...EI....D.............................. 153 Ref. ( accession no. AF269273)Roger130 80 ...............................V....DIK...T.............................. 153 Ref. ( accession no. AF269277)MS04 80 ...............................K....EI....D.............................. 153 Ref. ( accession no. DQ118378)pm39 80 ..........................K...N.D...DIK...EN.PTG......................... 153 Ref. ( accession no. AJ004876)Roger132 80 ..........................K...N.D...DIK...EN.PTG......................... 153 Ref. ( accession no. AF269278)

CS0401 79 ..........................K...D.V.........DG..NV......................... 152 3 II PG1CS0402 79 ..........................K...D.V.........DG..NV......................... 152 3 II PG2CS0508 79 ..........................K...D.V.........DG..NV......................... 152 3 IV PG7CS0515 79 ...............................A....DLK...T.............................. 152 16 V PG9CS0516 79 ...............................A....DLK...T.............................. 152 17 V PG9H7 79 ...............................K....DL....EK.A........................... 152 Ref. ( accession no. AF269276)

R19 78 ...............................A....DLK...T.............................. 151 Ref. ( accession no. AF269270)H5 78 ...............................A....DLK...T.............................. 151 Ref. ( accession no. AF269275)S45 78 ...............................V....DLK...T.............................. 151 Ref. ( accession no. AF269274)32XII 78 ...............................AD...KLE...E.............................. 151 Ref. ( accession no. AY687634)2V 78 ..........................K...D.A...EI....D.............................. 151 Ref. ( accession no. AY687633)13VII 78 .....................................K.....Q..DV......................... 151 Ref. ( accession no. AY687632)R22 78 ...............................K....EL....DT............................. 151 Ref. ( accession no. AF269271)

CS0501 77 ...................Q...H.......KP....T.....Q..DV......................... 150 4 III PG5CS0503 77 ...................Q..VH.......KP....T.....Q..DV.....................T... 150 6 III PG5CS0506 77 ..............................D.A..........N.ANV......................... 150 9 III PG6CS0511 77 ...................Q............A.....S....N..DV......................... 150 12 IV PG8CS0512 77 ...................Q............A.....S....N..DV......................... 150 13 IV PG8CS0513 77 ...................Q............A.....S....N..DV......................... 150 14 IV PG8CS0514 77 ...................Q............A.....S....N..DV......................... 150 15 IV PG8CS0518 77 ...................Q...H.......KD....Q.....N..DI......................... 150 19 V PG10CS0519 77 ...................Q...H.......KD....Q.....N..DI......................... 150 20 V PG10pm220 77 ...................Q...H.......KP....T.....Q..DV......................... 150 Ref. ( accession no. AJ005616)pmsh47 77 ...................Q...H.......KP....T.....Q..DV......................... 150 Ref. ( accession no. AJ004878)pm197 77 ...................Q...H.......KP....T.....Q..DV......................... 150 Ref. ( accession no. AJ004877)PCLH296 77 ...................Q...H.......KP....T.....Q..DV......................... 150 Ref. ( accession no. AJ004880)PCLH197 77 ...................Q...H.......KP....T.....Q..DV......................... 150 Ref. ( accession no. AJ440241)pmd1340 77 ...................Q...H.......KP....T.....Q..DV......................... 150 Ref. ( accession no. AJ004879)R24 77 ...................Q...H.......KD....Q.....N..DI......................... 150 Ref. ( accession no. AF269272)14V 77 ..............................D.A..........N.ANV......................... 150 Ref. ( accession no. AY687630)14VII 77 ..............................D.A..........N.ANV......................... 150 Ref. ( accession no. AY687631) *******************..*..**.***. *** .*** *. *********************.***

Clusters Strains Variable domain 2 Seq. types Farm no. Sample ID

A

B

C

D

Clusters Strains Variable domain 3 Seq. types Farm no. Sample ID

A

B

C

D

pm39 (AJ004876)

0.05 units

Roger132 (AF269278)

CS0507 (IV; PG7)

CS0401 (II; PG1)CS0402 (II; PG2)CS0508 (IV; PG7)

H7 (AF269276)

14V (AY687630)

14VII (AY687631)

CS0506 (III; PG6)CS0511 (IV: PG8)CS0514 (IV; PG8)CS0513 (IV; PG8)

CS0512 (IV; PG8)

pm220 (AJ005616)

CS0503 (III; PG5)

PCLH197 (AJ440241)

PCLH296 (AJ004880)

pm197 (AJ004877)

CS0501 (III; PG5)

pmd1340 (AJ004879)

pmsh47 (AJ004878)

R24 (AF269272)

CS0518 (V; PG10)CS0519 (V; PG10)

2V (AY687633)

R22 (AF269271)

13VII (AY687632)

H5 (AF269275)

R19 (AF269270)

S45 (AF269274)

32XII (AY687634)

CS0515 (V; PG9)CS0516 (V; PG9)

CS0504 (III; PG6)CS0505 (III; PG6)

R27 (AF269273)

CS0502 (III; PG5)CS0517 (V; PG9)

Roger130 (AF269277)

MS04 (DQ118378)

CS0510 (IV; PG7)

CS0302 (I; PG18)CS0304 (I; PG21)

CS0509 (IV; PG7)CS0303 (I; PG21)CS0301 (I; PG18)

C. trachomatis-LGV (L35605)

C. trachomatis-serovar-D (X62920)

C. trachomatis-serovar-C (AF352789)

C. muridarum-MoPn (X63409)

C. psittaci-MN Zhang (AF269281)

Human lymphogranuloma Worldwide

Human genital infection Worldwide

Mouse pneumonitis USA

Avian chlamydiosis Worldwide

Conjunctivitis Japan

Human eye infection Worldwide



Conjunctivitis JapanConjunctivitis Japan


Conjunctivitis Italy

Normal (jejunum) USA

Conjunctivitis JapanConjunctivitis JapanEnteritis (colon) USA



Normal (colon) Germany

Normal (intestine) USA

Enteritis (feces) USA

Conjunctivitis USA


Conjunctivitis USA



Conjunctivitis JapanResp. dis. (nasal mucosa) USA


Conjunctivitis JapanConjunctivitis JapanConjunctivitis JapanConjunctivitis JapanConjunctivitis JapanConjunctivitis Japan

Conjunctivitis JapanConjunctivitis JapanConjunctivitis Japan




Conjunctivitis USA

Normal (ilieum) USA

Disease/Source Geographical location

Bronchopneumonia Germany







Clusters

A

A

A

A

A

A

A

A

A

A

A

A

A

A

A

A

B

B

B

B

B

B

C

C

C

C

C

C

C

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

D

96.4%

99.9%

93.6%

100%

99.9%

90.2%

96.8%

100%

100%

90%

99.4%

100%

99.8%

100%

100%

100%

96%

100%

96.6%

Fig. 18. Neighbor-joining (NJ) phylogenetic tree, based on nucleotide sequences of VD2 and VD3 regions

of the ompA gene of different strains of C. suis and other Chlamydia spp. The strains detected in this study

are shown in bold letters along with farm and sample identity in parentheses. The corresponding genetic

clusters from A to D, isolation source/disease condition and geographical locations are shown against each

strain. The genetic distance is indicated in 0.05 unit bar. Bootstrap values are shown at respective nodes.

The C. psittaci is used as out-group and branch length is reduced to half.

96

suis among commercial piggeries. PCR based diagnosis is convenient and fast as isolation

of C. suis is considered as difficult due to antibiotic treatments or feed supplementations

(82, 178).

The 16S-23S rRNA intergenic spacer region and ompA are subjected to constant

systematic selective pressure and functional conservations and are ideal targets for

phylogenetic studies (20, 41). The genetic analysis of 16S-23S rRNA intergenic spacer

region and partial ompA gene revealed high genetic variations among C. suis strains. The

genetic variations in 16S-23S rRNA intergenic spacer region were less as compared to the

ompA gene. All the 9 variant 16S-23S rRNA intergenic spacer sequence types showed less

than 3% differences, whereas 20 ompA gene sequence variants detected from conjunctivitis

cases showed 22% genetic variation. The difference in the genetic variations between

ompA and 16S-23S rRNA intergenic spacer region may be due to the difference in

selective pressure at both loci associated with biphasic developmental cycle of chlamydiae

(20). In this study all the genetically variable strains of C. suis were detected from the

conjunctivitis cases but strains detected from gut and lung specimens are also reported to

have high degree of genetic variations (82).

Findings of this study are contradictory as compared to those of other chlamydiae

causing eye infections in animals, like feline conjunctivitis by C. felis (26) and inclusion

body conjunctivitis in guinea pigs by C. caviae (135). All the known strains of C. felis and

C. caviae have relatively conserved ompA loci (173, 227). However our findings correlate

with the situation in the human C. trachomatis strains, which have divergent ompA loci but

relatively conserved other portion of genome (20, 98, 189). Immunological selection might

promote such a process as change of MOMP peptide sequence, which might help the

chlamydiae to escape immune response.

97

According to the ompA gene composition, all genetically diverse strains were

divided into 4 groups. But the ompA gene based clusters of variant C. suis strains did not

show specific tissue tropism and disease association. These results are in accordance with

other studies suggesting no phylogenetic relationship among serovars that corresponds to

various pathobiotypes of C. trachomatis (20, 189). The C. suis strains of ophthalmic and

non-ophthalmic origin could not be differentiate neither in ompA gene nor 16S-23S rRNA

intergenic spacer region based phylogenetic analysis. Although serotypic variation has not

yet been studied in C. suis, genetic and biological variations have been reported among C.

suis strains (82, 95, 99, 183). The pathobiological significance of this high genetic

diversity is still not known.

Interestingly multiple genotypes of C. suis were involved in either individual

animal or a farm. The detection of multiple genotypes in ophthalmic swabs may also

indicate the repeated infection. Previous observations have also suggest that the severe

clinical symptoms and lesions seen in human trachoma are the results of repeated C.

trachomatis infection and are immunologically mediated (65, 130, 195). C. suis causes

mucopurulent keratitis/conjunctivitis under field conditions as observed in the present and

previous reports (165). However, the controlled experimental infection of gnotobiotic pigs

with H7 strain of C. suis (an eye isolate) was observed to be less severe than those of field

cases (163). It may be due to the use of single strain or no repetition of inoculations.

Besides the repetitive infection, other factors may be playing supportive role in disease

precipitation viz. poorly ventilated and dusty housing, houseflies (Musca domestica) and

other microbes like Mycoplasma hyorhinis, Klebsiella pneumoniae and Pasteurella sp.

(165).

In this study, mixed infection of C. suis along with other chlamydiae could not be

detect. It may be due to lack of sample diversity from non-ophthalmic origin. However, it

98

also suggests that ophthalmic infection is exclusively caused by C. suis. Although C. suis

has been isolated from porcine ocular samples in America and Italy only, there is no report

regarding role of chlamydiae in porcine conjunctivitis and keratoconjunctivitis in Japan or

any other Asian country. Further experimental studies are required to access the

pathogenicity of various C. suis strains and to understand biological significance of high

genetic diversity.

SUMMARY

Commercial piggeries with frequent ocular and reproductive problems were

etiologically investigated. Samples from 49 animals were collected from 5-pig farms

located in three prefectures of Japan from 2003 to 2005. Samples were screened

preliminarily with FAT and then followed by nested PCR tests. Chlamydia suis were

detected among 44.9% (22 out of 49) samples in all 5 farms by 16S-23S rRNA intergenic

spacer region based PCR and Chlamydia genus-specific PCR targeting ompA gene.

Whereas, all the samples were found negative by ompA gene based Chlamydophila genus

specific PCR. All the PCR positive samples were accrued from conjunctivitis cases only.

Total 9 C. suis strains with less than 3% nucleotide variation were detected by nucleotide

sequence analysis of 16S-23S rRNA intergenic spacer region, whereas, 20 C. suis strains

with up to 22% nucleotide variation were detected by analyzing ompA gene in VD2 and

VD3 regions. Multiple genotypes (up to 4 types) were found infecting either individual

animals or farms. This study revealed high prevalence of C. suis in porcine chlamydiosis

and association of genetically diverse, multiple genotypes in ophthalmic infection.

99

Molecular characterization of the Chlamydophila caviae strain OK135,

isolated from human genital tract infection, by analysis of some structural and

functional genes

INTRODUCTION

The chlamydiae cause a variety of diseases among human beings and animals (185,

188). Genital chlamydial infections are highly prevalent among adults and adolescents in

developing and developed countries (1, 89, 92, 204). Mostly Chlamydia trachomatis

(serovar D to K and L1-L3) and rarely Chlamydophila abortus are implicated in human

genital tract infections, manifested as salpingitis, cervicitis, pelvic inflammatory disease

(PID) and abortions.

Chlamydophila caviae, previously known as a guinea pig inclusion conjunctivitis

(GPIC) strain of Chlamydia psittaci, has been classified as a separate species under family

Chlamydiaceae based upon the 16S rRNA gene homology (43). C. caviae is a host specific

pathogen of guinea pigs and causes either self-limiting or asymptomatic inclusion

conjunctivitis among young (4-8 weeks old) guinea pigs (63, 101, 135). C. caviae and

guinea pig are important small animal experimental models to simulate the antigenic and

immune response of human chlamydial infections (118, 150-152). While C. caviae has no

previous history of infecting human beings, very recently, DNA of C. caviae was detected

in human, cat, rabbit eyes (114), and also in waste water samples (110), indicating the

possible wide ecobiological niche of C. caviae. Whole genome sequence of C. caviae

revealed many species-specific genes (153), although all the known 6 strains of C. caviae

CHAPTER-V

100

isolated from guinea pigs in different continents in different years were found to have

identical ompA gene sequences (227). In this chapter, characterization of a rare strain of C.

caviae, (OK135) was done, which was isolated from a woman with cervicitis by

Mastumoto et al. (unpublished work). Based on phylogenetic analysis of 16S rRNA, ompA

and groEL-1 genes, the OK135 strain was found to be genetically closely related to other

strains of C. caviae. This study highlights the ability of genetically diverse and non-

conventional chlamydiae to cause human genital tract infections and their probable escape

from detection due to use of diagnostic tests specific for conventional human chlamydial

species.


Case history: OK135 strain was isolated from a 20 years old female suffering

from severe cervicitis (Matsumoto et al., manuscript in preparation). The strain was

isolated and maintained subsequently in McCoy cell line. Preliminary examination of

strain was done by Giemsa and iodine staining and also by using chlamydial group specific

Mab in direct immunofluorescence test (Chlamydia FA, Denka Seiken Co. Ltd, Tokyo,

Japan). The results of iodine staining and C. trachomatis specific direct

immunofluorescence staining with Mab (MicroTrak; SYVA Co., Palo Alto, California,

USA) showed that OK135 strain is a non-conventional type of chlamydial species (122).


was used to extract DNA according to the instructions of manufacturer. After removing

cell debris by centrifugation at 1,000 ! g, partially purified elementary bodies were used

for DNA extraction. DNA was finally dissolved in 50 µl Tris-EDTA (TE) buffer pH 7.4

(100 mM Tris-HCl, pH 7.4 and 10 mM EDTA, pH 8.0) and stored at –30°C until used.

101

PCR amplification of 16S rRNA, ompA and groEL-1 genes: Overlapping DNA

fragments of 16S rRNA (1445 bp), ompA gene (1148 bp) and groEL-1 gene (1560 bp)

were amplified by single step PCR using pairs of primers as shown in Table 20. The 50 µl

PCR reaction mixture contained 0.20 µM of each forward and reverse primer, 250 µM of

each dATP, dTTP, dGTP, dCTP, 100 µM of Mg2+

in buffer and 2.5 units of TaKaRa Ex-

Taq (TaKaRa Bio Inc., Otsu, Japan) and 2.0 to 3.0 µg of template DNA. Thermocycling

conditions were adjusted according to primer sets and in all, 30 amplification cycles were

performed.

Cloning and sequencing of DNA fragments: The PCR amplified DNA fragments

of each gene were purified by QIAquick Gel Extraction kit (Qiagen, Hilden, Germany).

Then each DNA fragment was cloned in the pT7 blue vector (Novagen, Madison,

Wisconsin) by transforming E. coli DH10" strain by electroporation using Gene Pulser

cuvettes, 0.5 cm electrode gap (Bio Rad, Hercules, California). Clones containing DNA

insert were selected as in CHAPTER I (171). Total 3 to 5 clones with an expected size

DNA fragment insert were selected by using Premix Colony Direct PCR insert check kit

(Tyobo Co., Osaka, Japan) and taken for sequencing. Each DNA fragment was sequenced

by using thermo sequenase cycle sequencing Kit (USB Corporation, USA) using forward

(T7) and reverse (M13) universal fluorescent labeled primers using SEQ4!4 personal

sequencing system (Amersham Pharmacia Biotech).

Analysis of sequences and construction of phylogenetic trees: The obtained

sequences were assembled and edited using Genetyx-Mac/ATSQ 4.2.3 and Genetyx-Mac,

version 13.0.6 (SDC, Tokyo, Japan). For phylogenetic analysis, representative gene

sequences of 16S rRNA, ompA and groEL-1 genes of chlamydial species and other

bacteria associated with reproductive tract infections of animals and human were retrieved

from the DDBJ. Using ClustalX, version 1.83 (198), done the multiple alignments of the

102

Table 20. Primers used to amplify16S rRNA, ompA and groEL-1 genes.

Name Sequence Positionsa

Tm(°C)b

Amplicon

16S rRNA gene

CPR/P1F 5’ CTGAGAATTTGATCTTGGTTCAG 3’ 5-27 54-56 400 bp

CPR/P1R 5’ CGCTTCGTCAGACTTTCGTCCATT 3’ 404- 381 60

CPR/P2F 5’ CGTCTAGGCGGATTGAGAGATTG 3’ 291-313 60-62 418 bp

CPR/P2R 5’ CGCATTTCACCGCTACACGTGGAAT 3’ 708-684 60-62

CPR/P3F 5’ GCGTGTAGGCGGAAAGGAAAGTT 3’ 585-607 60-62 413 bp

CPR/P3R 5’ CCAGGTAAGGTTCTTCGCGTTGCAT 3’ 997-973 59-61

CPR/P4F 5’ CCGTGTCGTAGCTAACGCGTTAAGT 3’ 860-884 60-62 444 bp

CPR/P4R 5’ GGCTAGCTTTGAGGATTTGCTCCAT 3’ 1403-1279 59-61

CPR/P5F 5’ CGAGGATGACGTCAAGTCAGCAT 3’ 1191-1213 60-62 367 bp

CPR/P5R 5’ TGTCGACAA AGGAGGTGATCCAG 3’ 1557 –1537 55-57

ompA gene

M/0PF 5’ GCAAGTATAAGGAGTTATTGCTTG 3’ -275 to -252 54-56 352 bp

M/0PR 5’ CCTACAGGCAAGGCTTGTAAG 3’ 56-77 57-60

M/1PF 5’ CGGCATTATTGTTTGCCGCTAC 3’ 20-41 59 390 bp

M/1PR 5’ CGAAGCGATCCCAAATGTTTAAGGC 3’ 409-385 59-61

M/2PF 5’ CGCTTACGGAAGGCATATGCAAGATG 3’330-355 62 397 bp

M/2PR 5’ GTGAATCACAAATTGTGCTGGGCT 3’ 726-703 58-60

M/3PF 5’ CGTGGAGCTTTATGGGAATGTGGTTG 3’ 607-632 62 330 bp

M/3PR 5’ GAGCAATGCGGATAGTATCAGCATC 3’ 937-913 60-62

M/4PF 5’ GGCGTAAACTGGTCAAGAGCAAC 3’ 886-908 60-62 387 bp

M/4PR 5’ TCCCAGGTTCTGATAGCGGGACAA 1272-1246 61-63

groEL-1 gene

CVG/1PF 5’ GGCAGTTCTC AAGTAAGAGA AATC 3’ -66 to -43 56-58 392 bp

CVG/1PR 5’ GCAGTTACGTTTCTCAATCCTTC 3’ 326-304 55-58

CVG/2PF 5’ GCAGATAAAGCTGGTGATGGAAC 3’ 244-266 58-60 380 bp

CVG/2PR 5’ GGATTTGTAGAGAAGTAGCTGGAT 3’ 623-600 56-58

CVG/3PF 5’ CGAAACTGTCCTCGACGTTGT 3’ 549-569 57-60 405 bp

CVG/3PR 5’ CCTAACATAGCTAGAGTTGTGTTC 3’ 953-930 56-58

CVG/4PF 5’ GGTCAACTCATCAGCGAAGAG 3’ 892-912 57-60 436 bp

CVG/4PR 5’ GTGCTCCGATTTGCTCATCTTC 3’ 1306-1327 59

CVG/5PF 5’ CGCTGCATCCCTACTTTAGAAG 3’ 1261-1282 59 443 bp

CVG/5PF 5’ CCCTTTCTTCTCAAGATGGATG 3’ 1703-1682 57-59

a Based on 16S rRNA gene of Chlamydophila psittaci-Prk/GCP-1 strain (D85713), ompA gene of

C. psittaci-6BC strain (accession no. X56980) and groEL-1 gene of Chlamydophila caviae-GPIC

strain (accession no. AE015925). The negative values indicate the nucleotide positions upstream of

respective open reading frame. b Averages of melting temperatures of forward and reverse primer sets were used in the direct PCR

amplifications.

sequences. Phylogenetic analysis was done with programs available in the PHYLIP

(version 3.6a3; [http://evolution.genetics.washington.edu/phylip.html]). The distance

matrix between species was computed by DNADIST using Jukes-Cantor model (93) and

103

clustering was done using NJ method (170). The bootstrap values were calculated from a

consensus tree constructed by generating 1,000 random data sets using SEQBOOT (45). In

case of 16S rRNA gene, Parachlamydia acanthamoeba was taken as outgroup.

Extraction of chlamydial outer membrane complexes (COMC): Extraction of

chlamydial outer membrane complexes (COMC) was done as reported earlier by using 2%

sodium N-lauroyl sarcosine (Sarkosyl; Sigma) (27). The protein concentrations of the

extracts were determined by Bio-Rad protein assays Kit (Bio-Rad, CA). The proteins

profile of extracted COMC was visualized on sodium dodecyl sulfate polyacrilamide gel

electrophoresis (SDS-PAGE) (108).

RESULTS

The nucleotide sequences and resultant amino acid sequences of 16S rRNA, ompA

and groEL-1 genes were compared with sequences in the data bank by NCBI-BLAST

search (http://www.ncbi.nlm.nih.gov). The comparative phylogenetic analysis of 3

sequenced genes with previously reported chlamydial species is as follows:

16S rRNA gene: The 16S rRNA gene of OK135 strain was 100% homologous to

the C. caviae GPIC strain (accession no. AE015925), which was used by the Institute of

Genomic Reseach (TIGR) for whole genome sequencing. OK135 strain was also closely

related to another C. caviae GPIC strain (accession no. D85708 and ATCC No. VR-813,

isolated by E.S. Murray in 1964 from the conjunctiva of guinea pigs with conjunctivitis in

Massachusetts, USA), showing 99.7% (1509/1514) nucleotide homology (Fig. 19). The NJ

analysis of 16S rRNA gene also confirmed that the sequence of OK135 strain is identical

to that of C. caviae GPIC strain (accession no. AE015925). All the 3 strains of C. caviae

form a genetically distinct branch diversing from other Chamydophila spp. and Chlamydia

C. trachomatis- C/TW-3/OT (D85720)

C. trachomatis- L2/434/BU (U68443 )

C. trachomatis- D/UW-3/CX (AE001347)

C. trachomatis- E/UW-5/Cx (D85722)

C. suis- S45 (U73110)

C. muridarum- MoPn (AE002279)

C. pecorum- Koala type II (D85717)

C. pneumoniae- CWL029 (AE001668)

C. abortus- Ov/B577 (D85709)

C. psittaci- 6BC (U68447)

C. felis- Fe/145 (D85702)

C. caviae- GPIC (D85708)

C. caviae- GPIC (TIGR) (AE016997)

Parachlamydia acanthamoebae- Bn9 (Y07556)

C. psittaci- Prk/Daruma (U68447)

C. abortus- EBA (U76710)

99.4%

64.9%

100%

99.7%

51.7%55.3%

100%

94%

100%

83%

96.2%Bovine Abortion USA

Parrot Systemic infection Japan/India

Sheep Abortion USA

Parakeet Systemic infection USA

Guinea pig Conjunctivitis USA

Guinea pig Conjunctivitis USA

Cat Conjuctivitis USA

Koala Urogenital infection Australia

Human Pneumonitis Taiwan

Human Cervicitis USA

Human Trachoma Taiwan

Human Cervicitis USA

Human Lymphogranuloma USA

Pig Asymptomatic Austria

Mouse Pneumonitis USA

Acanthamoeba castellanii Germany

Human Cervicitis JapanC. caviae- OK135

0.05 units Species Disease conditions Location

Fig. 19. The NJ phylogenetic tree of the nucleotide sequence of 16S rRNA gene of the OK135 strain as compared to other chlamydialstrains of Chlamydiaceae family. The P. acanthamoebae was taken as out-group and its branch is shortened to half. The genetic dista--nce is shown as unit bar.

105

spp. This is supported by significantly high bootstrap value (Fig. 19).

ompA gene: The genetic composition of ompA gene, which has 4 genetically hyper

variable domains (VDs), was found to be identical in OK 135 and GPIC (TIGR) strains.

However, when compared to another strain-GPIC (sequence accession no. AF269282 and

isolate ATCC No. VR-813), 2 nucleotide differences were found. One at position 843 of

ORF (T base changed to C) with no change in amino acid residue and another at position

996 in VD4 (A base changed to T) with conversion of lysine residue to asparagines.

Interestingly, in another ompA gene sequence of C. caviae available in literature (226),

only single nucleotide polymorphism (SNP) at position 843 was observed. We also

sequenced promoter region of ompA gene up to 178 nucleotides upstream and found this

region to be conserved among all C. caviae strains. The NJ tree of ompA gene (without out

group) of all the nine species in Chlamydiaceae and all C. caviae strains, also provided

100% reliability support for the formation of distinct clade of C. caviae strains within the

family Chlamydiaceae (Fig. 20-A). The COMC composition of OK135 strain in SDS-

PAGE showed protein bands of MOMP and other proteins with identical sizes to the GPIC

(TIGR) strain.

groEL-1 gene: The GroEL-1 chaperonin of OK135 strain was 100% homologous

to other strains of C. caviae but the interspatial differences were observed. In the unrooted

NJ tree without out-group, the groEL-1 gene of other microbes commonly associated with

sexually transmitted diseases and members of order Chlamydials was found to be

phylogenetically distant (Fig. 20-B). The analysis of functional domains of this GroEL-1

chaperonin protein coded by this groEL-1 gene showed that all the functionally important

sites are conserved in family Chlamydiacae including this new OK135 strain (Table 21).

0.05 units

Neisseriae gonorrhoea(NG2095)

Parachlamydia acanthamoebae-

UWE25 (pc1180)

100%

C. pneumoniae-

C. trachomatis- serovar D (AJ783840) C. muridarum- MoPn

C. caviae- GPIC (AE015925)

(CTU52049)

M69217TWAR

100%100%

100%

Treponema pallidum (TP0030)

Streptococcus agalactiae (SAG2074)

Mycoplasma genitalium(MG392)

91.9%

99.8%

C. caviae-OK135

99.9%

(A)

(B)

0.05 units

C. muridarum- SFPD (U68437)

C. suis- PCLH197 (AJ440241)

C. trachomatis- c/TW3/OT (AF352789)

C. felis- Baker (AF269257)

C. psittaci- 6BC (X56980)

C. abortus- EBA (AF269256)

C. pecorum- LW613 (AJ440240)

C. pneumoniae- IOL-204 (M64064)

C. caviae- GPIC (AF269282)C. caviae- GPIC (TIGR) (AE015925)

C. caviae- OK135

C. caviae- GPIC (Ref)

100%

99.6%

95.4%

100%

Fig. 20-B. The NJ tree showing the phylogenetic distance in groEL-1 gene of OK135 strain (bold letters) vis-à-vis other C. caviae strains, chlamydial species and bacteria frequently in--volved in sexually transmitted infections.

Fig. 20-A. The NJ phylogenetic tree of the nucleotide sequence of the ompA gene of OK135strain (bold letters) and all 9 species of family Chlamydiaceae including all reported strainsof C. caviae.

Table 21. Multiple alignment of functional regions of GroEL-1 of OK135 strain vis-à-vis other chlamydial species and bacteria associated with reproductive diseases. The residues important for

polypeptide binding are boxed in solid line, for GroES contact in dashed line box and for ATPase activity in doted line box.

Amino acid residue positionsa

Organisms 87 88 89 90 91 150 151 152 199 201 203 204 234 237 259 263 264 265 361 383 405 406

Chlamydia trachomatis Asp Gly Thr Thr Thr Ile Ser Ala Tyr Ser Tyr Phe Leu Leu Leu Val Val Asp Asp Ala Ala Ala

Chlamydia muridarum Asp Gly Thr Thr Thr Ile Ser Ala Tyr Ser Tyr Phe Leu Leu Leu Val Val Asp Asp Ala Ala Ala

Chlamydophila pneumoniae Asp Gly Thr Thr Thr Ile Ser Ala Tyr Ser Tyr Phe Leu Leu Leu Val Val Asp Asp Ala Ala Ala

Chlamydophila caviae-OK135 Asp Gly Thr Thr Thr Ile Ser Ala Tyr Ser Tyr Phe Leu Leu Leu Val Val Asp Asp Ala Ala Ala

Chlamydophila caviae Asp Gly Thr Thr Thr Ile Ser Ala Tyr Ser Tyr Phe Leu Leu Leu Val Val Asp Asp Ala Ala Ala

Chlamydophila abortus Asp Gly Thr Thr Thr Ile Ser Ala Tyr Ser Tyr Phe Leu Leu Leu Val Val Asp Asp Ala Ala Ala

Chlamydophila abortus Asp Gly Thr Thr Thr Ile Ser Ala Tyr Ser Tyr Phe Leu Leu Leu Val Val Asp Asp Ala Ala Ala

Chlamydophila felis Asp Gly Thr Thr Thr Ile Ser Ala Tyr Ser Tyr Phe Leu Leu Leu Val Val Asp Asp Ala Ala Ala

Chlamydophila pecorum Asp Gly Thr Thr Thr Ile Ser Ala Tyr Ser Tyr Phe Leu Leu Leu Val Val Asp Asp Ala Ala Ala

Parachlamydia acanthamoeba Asp Gly Thr Thr Thr Ile Ser Ala Tyr Ser Tyr Phe Ile Leu Leu Val Val Asp Asp Ala Ala Ala

E. coli Asp Gly Thr Thr Thr Ile Ser Ala Tyr Ser Tyr Phe Leu Leu Leu Val Val Asp Asp Ala Ala Ala

Neisseriae gonorrhoea Asp Gly Thr Thr Thr Ile Ser Ala Tyr Ser Tyr Phe Leu Leu Leu Val Val Asp Asp Ala Ala Ala

Streptococcus agalactiae Asp Gly Thr Thr Thr Valb Ser Ser Tyr Ser Tyr Met Leu Leu Leu Val Leu Asp Asp Ala Ala Ala

Mycoplasma genitalium Asp Gly Thr Thr Thr Ile Ser Ser Tyr Ser Tyr Met Leu Leu Val Ala Val Asp Asp Gly Ala Ala

Treponema pallidum Asp Gly Thr Thr Thr Val Ser Ala Tyr Ser Tyr Phe Leu Leu Leu Val Val Asp Asp Ala Ala Ala

a The numbering of the amino acid residue positions is based on the E. coli GroEL-1.

b The substituted amino acid residue as compared to E. coli are shown in bold.

108

DISCUSSION

Chlamydiae have been reported in a variety of disease syndromes in human beings

and animals (185, 188). Among the Chlamydiales, C. trachomatis and C. pneumoniae are

established to be human pathogens. However, other Chlamydophila species can

occasionally infect human beings (113). In this chapter, a C. caviae strain, OK135, isolated

from a clinical human cervicitis case was identified and genetically characterized. C.

caviae usually cause self-limiting or asymptomatic ophthalmic infection in its natural host,

guinea pigs (63, 101, 135). Infection in human is unknown except a very recent report of

C. caviae DNA detection in human eye samples (114). The chlamydial cervicitis is usually

caused by C. trachomatis, serovars D to K, however, serovar E is reported to be common

(190).

Initially, 16S rRNA gene was analyzed for the phylogenetic identification of

OK135 strain as this gene formed the basis of recent classification of all the chlamydiae

(43). The 16S rRNA gene is reported to have species-specific conserved motifs in the order

Chlamydials. (43, 54, 149). OK135 strain was found to be genetically identical to GPIC

strain. Furthermore, the structural analysis of MOMP, which constitute majority of

chlamydial envelope and responsible for serological variations, was done (10, 50, 186,

187). Also genetic makeup of groEL-1 gene which encodes GroEL-1 (HSP 60) was

analyzed as these types of protein are recognized by the host Toll-like receptors (TLRs) as

part of innate defense system and implicated in diseases pathogenesis (23, 103).

The ompA gene locus of OK135 strain was identical to GPIC strain, and 1 to 2 SNP

were detected as compared to two other C. caviae sequences available in the data bank.

However, in other report, no allelic polymorphism was observed among 6 C. caviae strains

in partially sequenced (70%) ompA locus (227). All the 6 strains were identical to ompA

109

gene sequence of GPIC strain reported by Zhang et al. (226), which had a SNP at 843

position between VD3 and VD4 of ompA gene as compared to OK135 strain. Therefore,

all the C. caviae strains have relatively conserved ompA locus as compared to other

Chlamydia spp. and Chlamydophila spp., which show high polyporphism in ompA gene

(98, 189). Lack of high genetic variation among C. caviae strains as compared to other

chlamydiae with polymorphic ompA gene may be due to differences in host-pathogen

interaction and selective pressure exerted by host immune response. Furthermore, it has

also been observed that anti-MOMP serological response in C. caviae is not so protective

in experimental ophthalmic and genital experimental infections (13, 203). This is similar to

the non-prominent anti-MOMP immune response shown by C. pnemoniae that also lack

polymorphism in ompA gene locus (28). The C. pneumoniae infection are widely prevalent

among human population (106). Although the C caviae GPIC has been studied extensively

as surrogate experimental strain to understand pathogenesis and immune responses in

human genital infections (150, 215), the C. caviae infection among human are uncommon

or rather unknown. In the present case, while the circumstantial evidences of contacting C.

caviae by the said patient from primary host (guinea pig) are lacking, the possible reason

for switching of host species may be due to lack of protective immunity in advance or

cross protection among human to C. caviae infection. Another possible source of infection

may be still unknown host or carrier of C. caviae as the recent reports are also indicating

possible wider ecobiological niche (110, 114).

It was also observed that functional sites in GroEL-1 protein (HPS 60) coded by

groEL-1 gene are conserved among all the members of Chlamydiaceae family, it can be

speculated that chlamydiae may exhibit a similar disease pathogenesis or ability to infect

multiple host species. Therefore, diagnosis of aberrant chlamydial species in various

clinical manifestations is important. These types of infection may exist in human

110

population but remained undiagnosed due to use of species specific diagnostic methods in

chlamydial reproductive tract infections viz. PCR or LCR Kit are mostly targeted at 7.5-kb

plasmid or C. trachomatis specific monoclonal antibody based fluorescent tests and in such

cases the clinical diagnosis of C. caviae can be bypassed (121). It is also observed that

severity of clinical manifestation and inflammation of urogenital chlamydia infections is

strongly influenced by the number of chlamydiae present in the genital tract rather than the

infecting serovar of C. trachomatis (61). Hence, undiagnosed infection with such rare

chlamydial species can proliferate in genital tract can lead to pelvic inflammatory disease

(PID) and its subsequent complications like salpingitis and tubal factor infertility, similar

to those occurred by other genital chlamydial species. This study also suggests need for

development of new diagnostic tools for chlamydial reproductive infections, encompassing

broader Chlamydiaceae group.

SUMMARY

Genital tract infections by different serovars of C. trachomatis are reported to be

alarmingly higher in recent studies. Occasionally C. abortus also causes human

reproductive disorders. The C. caviae is a highly host specific species and causes

conjunctivitis and other eye infections in guinea pigs. In our study, we characterize a C.

caviae strain (OK135) isolated from a woman with acute cervicitis, by sequencing 16S

rRNA, ompA and groEL-1 genes. The isolate was consequently confirmed as C. caviae and

phylogenetically closely related to other strains of C. caviae. The ompA gene had 100%

homology to GPIC strain of C. caviae used for genome project by TIGR, but has 1 to 2

nucleotide substitution in the coding region as compared to other already reported C.

caviae strains. The COMC extracted by sarkosyl detergent showed 38.5 kDa size of

111

MOMP along with other proteins, similar to those of GPIC strain. The groEL-1 and 16S

rRNA genes also showed 100% homologous to those of GPIC strains. Therefore, the

OK135 strain of C. caviae appears to be closely related to GPIC strain. This report

highlights the ability of non-conventional chlamydiae to cause human infections without

many genetic and subsequent structural and physiological adjustments. It suggests the need

for broader and not species-specific diagnostic approach for human chlamydial infections.

112

Chlamydiae are the obligate intracellular bacterial pathogens of animals and human

beings. They are responsible for a diverse range of diseases in birds and mammals

including humans. C. trachomatis and C. pneumoniae are established to be human

pathogens. Animal chlamydial infections are caused by C. psittaci, C. abortus, C. suis, C.

muridarum, C. pecorum, C. felis and C. caviae. Various studies have shown natural

prevalence of genetically diverse strains of chlamydiae within each species. C. psittaci is

mainly prevalent among avian species and causes either inapparent or clinical infection of

varying severity. C. psittaci is also considered to be potentially pathogenic to in-contact

mammalian hosts including human beings.

This thesis describes the molecular epidemiology, genetic diversity and virulence of

C. psittaci strains, which were naturally distributed among diverse types of avian fauna, in

order to compare the risk to the health of avian and mammalian fauna as well as human

beings. This study was also intended to detect the chlamydiae in some diseased animals

and humans, as different chlamydial species were detected recently from previously known

disease syndromes.

In the molecular epidemiological study to investigate the diversity and natural

distribution of C. psittaci, total 1,147 samples from 11 avian orders including 53 genera

and 113 species of feral and captive birds were examined using ompA gene based nested

PCR. Three types of chlamydiae including C. psittaci (94.12%), C. abortus (4.41%) and

unknown Chlamydophila sp. (1.47%) were identified among 68 birds, which includes 59

from Psittaciformes, 8 from Ciconiiformes and 1 from Passeriformes orders. Based on

nucleotide sequence variations in the VD2 region of ompA gene, all 64 detected C. psittaci

strains were grouped into 4 genetic clusters. Clusters I, II, III and IV were detected from

CONCLUSION

113

57.35%, 19.12%, 10.29% and 7.35% samples, respectively. A single strain of unknown

Chlamydophila sp. was found to be phylogenetically intermediate between Chlamydophila

species of avian and mammalian origins. Therefore, it was concluded that, though various

genetically diverse chlamydiae may have caused avian chlamydiosis, only a few C. psittaci

strains were highly prevalent and frequently associated with clinical/subclinical infections.

All the 46 known and genetically diverse strains of C. psittaci, detected in the

present and previous studies from various avian and mammalian hosts including human

beings were genetically analyzed to evaluate the phylogenetic relationships, pathobiotic

implications and to improve the strain categorization. The ompA gene loci of 11 new C.

psittaci strains along with previously known 35 strains were analyzed. 16S rRNA gene of

two highly diverse strains CPX0308 and Daruma was also studied. On the basis of the

conserved region of ompA gene, 4 lineages of C. psittaci strains with genetic distance of

less than 0.1 (dissimilarity less than 10%) were noted. The C. psittaci strains of 4 lineages,

with genetic distance of less than 0.05 (dissimilarity less than 5%) were subdivided into 8

genetic clusters. The ompA gene loci of C. psittaci strains of lineage-1 were relatively

conserved and these strains were frequently involved in infection of human (psittacosis)

and other mammalian species. Lineage-2 strains are genetically diverse and divided into 5

genetic clusters. Lineage-3 and 4 represented by WC and CPX0308 strains, respectively,

are genetically more close to Chlamydophila spp. causing infection among mammalian

hosts. The proposed scheme classifies diverse C. psittaci strains according to the genetic

variations and phylogenetic relationships. It may also be useful to classify even uncultured

C. psittaci strains detected in clinical diagnosis.

Further to evaluate the degree of virulence of C. psittaci strains predominantly

responsible for avian chlamydiosis and human psittacosis, BALB/c mice were used as

experimental infection model. Four representative C. psittaci strains: No!e, MN, Mat116

114

and Borg were intranasally inoculated at dose rate of 0.5 IFU, 5 IFU, 50 IFU, 5!102 IFU,

5!103 IFU and 5!10

4 IFU per gram body weight. The LD50 of Nosé, MN, Mat116 and

Borg strains were 3.1!103, 19!10

3, 12!10

3 and 6.3!10

3 IFU/gm body weight, respectively.

The intensity of clinical signs against the infectious doses of 5!103 and 5!10

4 IFU/gm

body weight was the same among the 4 strains, however, at 50 and 5!102 IFU/gm body

weight doses, the MN strain showed milder clinical signs. The animals incubated with the

MN strain at higher doses showed less variation in the relative body weights as compared

to those inoculated with the Nosé, Mat116 and Borg strains. Among the 4 strains, complete

recovery and clearance of chlamydial infection was verified at 21 days post infection, as no

chlamydial DNA and/or chlamydial IFU was detected in lung, liver and spleen tissues.

Chlamydia specific IgG immune response was found to be dose dependent. This study

indicated that the C. psittaci strains frequently associated with avian and human psittacosis

cases were highly virulent to mice and that the immune responses lead to variable degrees

of protection. Our murine lung infection based model can be used for further molecular

virulence analysis studies.

The chlamydial etiological investigation in ophthalmic and reproductive diseases of

animals and human showed some interesting results. The detected chlamydial species and

strains were genetically characterized by multilocus sequence analysis.

Five commercial piggeries, in three prefectures of Japan, with frequent ocular and

reproductive problems were etiologically investigated. Samples from 49 animals were

collected and screened preliminarily with FAT and then followed by nested PCR tests. C.

suis were detected in 44.9% (22 out of 49) samples in all 5 farms by 16S-23S rRNA

intergenic spacer region based PCR and Chlamydia genus-specific PCR targeting ompA

gene. Whereas, all the samples were found to be negative by ompA gene based

Chlamydophila genus specific PCR. All the PCR positive samples were accrued from

115

conjunctivitis cases only. Total 9 C. suis strains with less than 3% nucleotide variation

were detected by nucleotide sequence analysis of 16S-23S rRNA intergenic spacer region,

whereas, 20 C. suis strains with up to 22% of nucleotide variation were detected by

analyzing VD2 and VD3 regions of the ompA gene. Multiple genotypes (up to 4 types)

were found infecting either individual animals or farms. This study revealed high

prevalence of C. suis in porcine chlamydiosis and association of genetically diverse,

multiple genotypes in ophthalmic infection.

Human genital tract infections by different serovars of C. trachomatis are reported

to be alarmingly higher in recent epidemiological studies. Occasionally C. abortus also

causes human reproductive disorders, whereas, C. caviae is a highly host specific species

and causes conjunctivitis and other eye infections in guinea pigs. In our study, we

characterize a C. caviae strain (OK135) isolated from a woman with acute cervicitis, by

sequencing 16S rRNA, ompA and groEL-1 genes. The isolate was confirmed as C. caviae

and phylogenetically closely related to other previously reported strains of C. caviae. The

ompA, groEL-1 and 16S rRNA genes showed 100% homology to GPIC strain of C. caviae.

This report highlights the ability of non-conventional chlamydiae to cause human

infections without many genetic and subsequent structural and physiological adjustments.

It suggests the need for broader and not species-specific diagnostic approach in human

chlamydial infections.

The results of this study showed that, though C. psittaci is widely distributed

among avian fauna, only a few strains/genotypes are epidemiologically successful and are

highly virulent to in-contact animals or human beings. In general chlamydiae may be

involved in many animal or human disease conditions but either have not yet been

investigated in detail or have escaped from clinical diagnosis due to use of very specific

diagnostic approaches.

116

1. Adams, E. J., A. Charlett, W. J. Edmunds, and G. Hughes. 2004. Chlamydia

trachomatis in the United Kingdom: a systematic review and analysis of prevalence

studies. Sex. Transm. Infect. 80:354-362.

2. Alexander, E. R., S. P. Wang, and J. T. Grayston. 1967. Further classification of

TRIC agents from ocular trachoma and other sources by the mouse toxicity

prevention test. Am. J. Ophthalmol. 63:Suppl:1469-1478.

3. Andersen, A. A. 1991. Comparison of avian Chlamydia psittaci isolates by

restriction endonuclease analysis and serovar-specific monoclonal antibodies. J.

Clin. Microbiol. 29:244-249.

4. Andersen, A. A. 1991. Serotyping of Chlamydia psittaci isolates using serovar-

specific monoclonal antibodies with the microimmunofluorescence test. J. Clin.

Microbiol. 29:707-711.

5. Andersen, A. A., J. E. Grimes, and H. L. Shivaprasad. 1998. Serotyping of

Chlamydia psittaci isolates from ratites. J. Vet. Diagn. Invest. 10:186-188.

6. Andersen, A. A., and J. P. Tappe. 1989. Genetic, immunologic, and pathologic

characterization of avian chlamydial strains. J. Am. Vet. Med. Assoc. 195:1512-

1516.

7. Andersen, A. A., and D. Vanrompay. 2000. Avian chlamydiosis. Rev. Sci. Tech.

19:396-404.

8. Andersen, A. A., and D. Vanrompay. 2003. Avian Chlamydiosis (psittacosis,

ornithosis), p. 863-879. In Y. M. Saif, H. J. Barnes, J. R. Glisson, A. M. Fadly, L.

R. McDougald, and D. E. Swayne (ed.), Diseases of Poultry, 11 ed. Iowa State

Press, A Blackwell Publishing Company, Ames Iowa.

9. Anderson, D. C., P. A. Stoesz, and A. F. Kaufmann. 1978. Psittacosis outbreak in

employees of a turkey-processing plant. Am. J. Epidemiol. 107:140-148.

REFERENCES

117

10. Baehr, W., Y. X. Zhang, T. Joseph, H. Su, F. E. Nano, K. D. Everett, and H. D.

Caldwell. 1988. Mapping antigenic domains expressed by Chlamydia trachomatis

major outer membrane protein genes. Proc. Natl. Acad. Sci. U. S. A. 85:4000-4004.

11. Banks, J., B. Eddie, M. Sung, N. Sugg, J. Schachter, and K. F. Meyer. 1970. Plaque

reduction technique for demonstrating neutralizing antibodies for Chlamydia.

Infect. Immun. 2:443-447.

12. Batteiger, B. E., P. M. Lin, R. B. Jones, and B. J. Van Der Pol. 1996. Species-,

serogroup-, and serovar-specific epitopes are juxtaposed in variable sequence

region 4 of the major outer membrane proteins of some Chlamydia trachomatis

serovars. Infect. Immun. 64:2839-2841.

13. Batteiger, B. E., and R. G. Rank. 1987. Analysis of the humoral immune response

to chlamydial genital infection in guinea pigs. Infect. Immun. 55:1767-1773.

14. Bedson, S. P., G. T. Western, and S. L. Simpson. 1930. Observations on the

aetiology of psittacosis. Lancet. i:235-236.

15. Bell, S. D., Jr., J. C. Snyder, and E. S. Murray. 1959. Immunization of mice against

toxic doses of homologous elementary bodies of trachoma. Science. 130:626-627.

16. Bernstein-Hanley, I., Z. R. Balsara, W. Ulmer, J. Coers, M. N. Starnbach, and W.

F. Dietrich. 2006. Genetic analysis of susceptibility to Chlamydia trachomatis in

mouse. Genes Immun. 7:122-129.

17. Bovarnick, M. R., J. C. Miller, and J. C. Snyder. 1950. The influence of certain

salts, amino acids, sugars, and proteins on the stability of rickettsiae. J. Bacteriol.

59:509-522.

18. Bracewell, C. D., and B. J. Bevan. 1986. Chlamydiosis in birds in Great Britain. 1.

Serological reactions to chlamydia in birds sampled between 1974 and 1983. J.

Hyg. (Lond.). 96:447-451.

19. Browning, G. F. 2004. Is Chlamydophila felis a significant zoonotic pathogen?

Aust. Vet. J. 82:695-696.

118

20. Brunelle, B. W., and G. F. Sensabaugh. 2006. The ompA gene in Chlamydia

trachomatis differs in phylogeny and rate of evolution from other regions of the

genome. Infect. Immun. 74:578-585.

21. Brunham, R. C., C. Kuo, and W. J. Chen. 1985. Systemic Chlamydia trachomatis

infection in mice: a comparison of lymphogranuloma venereum and trachoma

biovars. Infect. Immun. 48:78-82.

22. Buendia, A. J., L. Del Rio, N. Ortega, J. Sanchez, M. C. Gallego, M. R. Caro, J. A.

Navarro, F. Cuello, and J. Salinas. 2002. B-cell-deficient mice show an exacerbated

inflammatory response in a model of Chlamydophila abortus infection. Infect.

Immun. 70:6911-6918.

23. Bulut, Y., E. Faure, L. Thomas, H. Karahashi, K. S. Michelsen, O. Equils, S. G.

Morrison, R. P. Morrison, and M. Arditi. 2002. Chlamydial heat shock protein 60

activates macrophages and endothelial cells through Toll-like receptor 4 and MD2

in a MyD88-dependent pathway. J. Immunol. 168:1435-1440.

24. Bush, R. M., and K. D. Everett. 2001. Molecular evolution of the Chlamydiaceae.

Int. J. Syst. Evol. Microbiol. 51:203-220.

25. Buxton, D. 1986. Potential danger to pregnant women of Chlamydia psittaci from

sheep. Vet. Rec. 118:510-511.

26. Cai, Y., H. Fukushi, S. Koyasu, E. Kuroda, T. Yamaguchi, and K. Hirai. 2002. An

etiological investigation of domestic cats with conjunctivitis and upper respiratory

tract disease in Japan. J. Vet. Med. Sci. 64:215-219.

27. Caldwell, H. D., J. Kromhout, and J. Schachter. 1981. Purification and partial

characterization of the major outer membrane protein of Chlamydia trachomatis.

Infect. Immun. 31:1161-1176.

28. Campbell, L. A., C. C. Kuo, S. P. Wang, and J. T. Grayston. 1990. Serological

response to Chlamydia pneumoniae infection. J. Clin. Microbiol. 28:1261-1264.

29. Carella, G., L. Marra, and T. Vallot. 1996. Hepatic psittacosis: a case of liver

abnormality diagnosed by ultrasonography. Presse Med. 25:197-198.

119

30. Chahota, R., R. C. Katoch, and M. K. Batta. 1997. Prevalence of Chlamydia

psittaci among feral birds in Himachal Pradesh, India. J. Appl. Anim. Res. 12:89-

94.

31. Chahota, R., R. C. Katoch, and V. B. Joshi. 1997. Seroprevalence of Chlamydia

psittaci in domestic poultry and wild birds. Indian J. Poult. Sci. 32:67-71.

32. Chahota, R., R. C. Katoch, S. P. Singh, S. Verma, and A. Mahajan. 2000.

Concurrent outbreak of chlamydiosis and aflatoxicosis among chickens in

Himachal Pradesh, India. VETERINARSKI ARHIV. 70:207-213.

33. Chahota, R., H. Ogawa, Y. Mitsuhashi, K. Ohya, T. Yamaguchi, and H. Fukushi.

2006. Genetic diversity and epizootiology of Chlamydophila psittaci prevalent

among the captive and feral avian species based on VD2 region of ompA gene.

Microbiol. Immunol. 50:663-678.

34. Coles, A. C. 1930. Micro-organisms in psittacosis. Lancet. i:1011-1012.

35. Cox, H. U., P. G. Hoyt, R. P. Poston, T. G. Snider, 3rd, T. X. Lemarchand, and K.

L. O'Reilly. 1998. Isolation of an avian serovar of Chlamydia psittaci from a case

of bovine abortion. J. Vet. Diagn. Invest. 10:280-282.

36. Crosse, B. A. 1990. Psittacosis: a clinical review. J. Infect. 21:251-259.

37. De Schrijver, K. 1998. A psittacosis outbreak in customs officers in Antwerp

(Belgium). Bull. Inst. Marit. Trop. Med. Gdynia. 49:97-99.

38. Denamur, E., C. Sayada, A. Souriau, J. Orfila, A. Rodolakis, and J. Elion. 1991.

Restriction pattern of the major outer-membrane protein gene provides evidence for

a homogeneous invasive group among ruminant isolates of Chlamydia psittaci. J.

Gen. Microbiol. 137 (Pt 11): 2525-2530.

39. Eidson, M. 2002. Psittacosis/avian chlamydiosis. J. Am. Vet. Med. Assoc.

221:1710-1712.

40. Everett, K. D. 2000. Chlamydia and Chlamydiales: more than meets the eye. Vet.


120

41. Everett, K. D., and A. A. Andersen. 1997. The ribosomal intergenic spacer and

domain I of the 23S rRNA gene are phylogenetic markers for Chlamydia spp. Int. J.

Syst. Bacteriol. 47:461-473.

42. Everett, K. D., A. A. Andersen, M. Plaunt, and T. P. Hatch. 1991. Cloning and

sequence analysis of the major outer membrane protein gene of Chlamydia psittaci

6BC. Infect. Immun. 59:2853-2855.

43. Everett, K. D., R. M. Bush, and A. A. Andersen. 1999. Emended description of the

order Chlamydiales, proposal of Parachlamydiaceae fam. nov. and Simkaniaceae

fam. nov., each containing one monotypic genus, revised taxonomy of the family

Chlamydiaceae, including a new genus and five new species, and standards for the

identification of organisms. Int. J. Syst. Bacteriol. 49 Pt 2:415-440.

44. Everett, K. D., L. J. Hornung, and A. A. Andersen. 1999. Rapid detection of the

Chlamydiaceae and other families in the order Chlamydiales: three PCR tests. J.

Clin. Microbiol. 37:575-580.

45. Felsenstein, J. 1985. Confidence limits on phylogenies: an approach using the

bootstrap. Evolution. 39:783-791.

46. Felsenstein, J., and G. A. Churchill. 1996. A hidden Markov model approach to

variation among sites in rate of evolution. Mol. Biol. Evol. 13:93-104.

47. Fitch, W. M., E. M. Peterson, and L. M. de la Maza. 1993. Phylogenetic analysis of

the outer-membrane-protein genes of Chlamydiae, and its implication for vaccine

development. Mol. Biol. Evol. 10:892-913.

48. Fox, J. G., H. F. Stills, B. J. Paster, F. E. Dewhirst, L. Yan, L. Palley, and K.

Prostak. 1993. Antigenic specificity and morphologic characteristics of Chlamydia

trachomatis, strain SFPD, isolated from hamsters with proliferative ileitis. Lab.

Anim. Sci. 43:405-410.

49. Francis, T. J., and T. P. Magill. 1938. An unidentified virus producing acute

meningitis and pneumonia in experimental animals. J. Exp. Med. 68:147-160.

121

50. Fukushi, H., and K. Hirai. 1988. Immunochemical diversity of the major outer

membrane protein of avian and mammalian Chlamydia psittaci. J. Clin. Microbiol.

26:675-680.

51. Fukushi, H., and K. Hirai. 1989. Genetic diversity of avian and mammalian

Chlamydia psittaci strains and relation to host origin. J. Bacteriol. 171:2850-2855.

52. Fukushi, H., and K. Hirai. 1992. Proposal of Chlamydia pecorum sp. nov. for

Chlamydia strains derived from ruminants. Int. J. Syst. Bacteriol. 42:306-308.

53. Fukushi, H., and K. Hirai. 1993. Chlamydia pecorum--the fourth species of genus

Chlamydia. Microbiol. Immunol. 37:516-522.

54. Fukushi, H., and K. Hirai. 1993. Restriction fragment length polymorphisms of

rRNA as genetic markers to differentiate Chlamydia spp. Int. J. Syst. Bacteriol.

43:613-617.

55. Fukushi, H., K. Itoh, Y. Ogawa, Y. Hayashi, M. Kuzuya, K. Hirai, and S.

Shimakura. 1983. Isolation and serological survey of Chlamydia psittaci in feral

pigeons from Japan. Nippon Juigaku Zasshi. 45:847-848.

56. Fukushi, H., K. Nojiri, and K. Hirai. 1987. Monoclonal antibody typing of

Chlamydia psittaci strains derived from avian and mammalian species. J. Clin.

Microbiol. 25:1978-1981.

57. Fukushi, H., H. Ogawa, N. Minamoto, A. Hashimoto, K. Yagami, H. Tamura, S.

Shimakura, and K. Hirai. 1985. Seroepidemiological surveillance of Chlamydia

psittaci in cats and dogs in Japan. Vet. Rec. 117:503-504.

58. Fukushi, H., H. Ogawa, M. Toshimichi, O. Yasuyuki, S. Seigo, and K. Hirai. 1985.

Chlamydial complement fixing antibodies in cows, horses and pigs from 1980 to

1983. Res. Bull. Fac. Agr. Gifu Univ. 50:259-263.

59. Gaydos, C. A., T. C. Quinn, and J. J. Eiden. 1992. Identification of Chlamydia

pneumoniae by DNA amplification of the 16S rRNA gene. J. Clin. Microbiol.

30:796-800.

122

60. Geens, T., A. Desplanques, M. Van Loock, B. M. Bonner, E. F. Kaleta, S.

Magnino, A. A. Andersen, K. D. Everett, and D. Vanrompay. 2005. Sequencing of

the Chlamydophila psittaci ompA gene reveals a new genotype, E/B, and the need

for a rapid discriminatory genotyping method. J. Clin. Microbiol. 43:2456-2461.

61. Geisler, W. M., R. J. Suchland, W. L. Whittington, and W. E. Stamm. 2001.

Quantitative culture of Chlamydia trachomatis: relationship of inclusion-forming

units produced in culture to clinical manifestations and acute inflammation in

urogenital disease. J. Infect. Dis. 184:1350-1354.

62. Girjes, A. A., F. N. Carrick, and M. F. Lavin. 1994. Remarkable sequence

relatedness in the DNA encoding the major outer membrane protein of Chlamydia

psittaci (koala type I) and Chlamydia pneumoniae. Gene. 138:139-142.

63. Gordon, F. B., E. Weiss, A. L. Quan, and H. R. Dressler. 1966. Observations on

guinea pig inclusion conjunctivitis agent. J. Infect. Dis. 116:203-207.

64. Grayston, J. T., C. C. Kuo, L. A. Campbell, and S. P. Wang. 1989. Chlamydia

pneumoniae sp. nov. for Chlamydia sp. strain TWAR. Int. J. Syst. Bacteriol. 39:88-

90.

65. Grayston, J. T., S. P. Wang, L. J. Yeh, and C. C. Kuo. 1985. Importance of

reinfection in the pathogenesis of trachoma. Rev. Infect. Dis. 7:717-725.

66. Green, S. T., N. W. Hamlet, L. Willocks, K. Sherry, D. H. Kennedy, C. E. Frew,

and D. Carrington. 1990. Psittacosis presenting with erythema-marginatum-like

lesions--a case report and a historical review. Clin. Exp. Dermatol. 15:225-227.

67. Gregory, D. W., and W. Schaffner. 1997. Psittacosis. Semin. Respir. Infect. 12:7-

11.

68. Gresham, A. C., C. E. Dixon, and B. J. Bevan. 1996. Domiciliary outbreak of

psittacosis in dogs: potential for zoonotic infection. Vet. Rec. 138:622-623.

69. Grimes, J. E., and F. D. Clark. 1986. Chlamydia psittaci infections of pet and other

birds in the United States in 1984. J. Infect. Dis. 153:374-375.

123

70. Haag-Wackernagel, D., and H. Moch. 2004. Health hazards posed by feral pigeons.

J. Infect. 48:307-313.

71. Hahn, D. L., R. W. Dodge, and R. Golubjatnikov. 1991. Association of Chlamydia

pneumoniae (strain TWAR) infection with wheezing, asthmatic bronchitis, and

adult-onset asthma. JAMA. 266:225-230.

72. Harris, R. L., and T. W. Williams, Jr. 1985. “Contribution to the Question of

Pneumotyphus”: a discussion of the original article by J. Ritter in 1880. Rev. Infect.

Dis. 7:119-122.

73. Harrison, H. R., S. M. Lee, and D. O. Lucas. 1982. Chlamydia trachomatis

pneumonitis in the C57BL/KsJ mouse: pathologic and immunologic features. J.

Lab. Clin. Med. 100:953-962.

74. Hartley, J. C., S. Stevenson, A. J. Robinson, J. D. Littlewood, C. Carder, J.

Cartledge, C. Clark, and G. L. Ridgway. 2001. Conjunctivitis due to

Chlamydophila felis (Chlamydia psittaci feline pneumonitis agent) acquired from a

cat: case report with molecular characterization of isolates from the patient and cat.

J. Infect. 43:7-11.

75. Hatch, T. P. 1999. Developmental Biology, p. 29-67. In R. S. Stephens (ed.),

Chlamydia: intracellular biology, pathogenesis and immunity. American Society

for Microbiology, Washington, DC.

76. Heddema, E. R., S. Ter Sluis, J. A. Buys, C. M. Vandenbroucke-Grauls, J. H. van

Wijnen, and C. E. Visser. 2006. Prevalence of Chlamydophila psittaci in fecal

droppings from feral pigeons in Amsterdam, The Netherlands. Appl. Environ.

Microbiol. 72:4423-4425.

77. Heddema, E. R., E. J. van Hannen, B. Duim, B. M. de Jongh, J. A. Kaan, R. van

Kessel, J. T. Lumeij, C. E. Visser, and C. M. Vandenbroucke-Grauls. 2006. An

outbreak of psittacosis due to Chlamydophila psittaci genotype A in a veterinary

teaching hospital. J. Med. Microbiol. 55:1571-1575.

124

78. Herrmann, B., H. Persson, J. K. Jensen, H. D. Joensen, M. Klint, and B. Olsen.

2006. Chlamydophila psittaci in Fulmars, the Faroe Islands. Emerg. Infect. Dis.

12:330-332.

79. Herrmann, B., R. Rahman, S. Bergstrom, J. Bonnedahl, and B. Olsen. 2000.

Chlamydophila abortus in a Brown skua (Catharacta antarctica lonnbergi) from a

subantarctic island. Appl. Environ. Microbiol. 66:3654-3656.

80. Hewinson, R. G., S. E. Rankin, B. J. Bevan, M. Field, and M. J. Woodward. 1991.

Detection of Chlamydia psittaci from avian field samples using the PCR. Vet. Rec.

128:129-130.

81. Hill, J. E., S. H. Goh, D. M. Money, M. Doyle, A. Li, W. L. Crosby, M. Links, A.

Leung, D. Chan, and S. M. Hemmingsen. 2005. Characterization of vaginal

microflora of healthy, nonpregnant women by chaperonin-60 sequence-based

methods. Am. J. Obstet. Gynecol. 193:682-692.

82. Hoelzle, L. E., G. Steinhausen, and M. M. Wittenbrink. 2000. PCR-based detection

of chlamydial infection in swine and subsequent PCR-coupled genotyping of

chlamydial omp1-gene amplicons by DNA-hybridization, RFLP-analysis, and

nucleotide sequence analysis. Epidemiol. Infect. 125:427-439.

83. Holland, S. M., C. A. Gaydos, and T. C. Quinn. 1990. Detection and differentiation

of Chlamydia trachomatis, Chlamydia psittaci, and Chlamydia pneumoniae by

DNA amplification. J. Infect. Dis. 162:984-987.

84. Hotzel, H., A. Berndt, F. Melzer, and K. Sachse. 2004. Occurrence of

Chlamydiaceae spp. in a wild boar (Sus scrofa L.) population in Thuringia

(Germany). Vet. Microbiol. 103:121-126.

85. Hughes, C., P. Maharg, P. Rosario, M. Herrell, D. Bratt, J. Salgado, and D.

Howard. 1997. Possible nosocomial transmission of psittacosis. Infect. Control

Hosp. Epidemiol. 18:165-168.

86. Hughes, E. S., K. M. Shaw, and R. H. Ashley. 2001. Mutagenesis and functional

reconstitution of chlamydial major outer membrane proteins: VS4 domains are not

125

required for pore formation but modify channel function. Infect. Immun. 69:1671-

1678.

87. Hyde, S. R., and K. Benirschke. 1997. Gestational psittacosis: case report and

literature review. Mod. Pathol. 10:602-607.

88. Illner, V. F. 1960. Zur Frage der Ubertragung des Ornithosevirus durch das Ei.

Monatsh. Veterinarinaermed. 17:116-117.

89. Imai, H., H. Shinohara, H. Nakao, H. Tsukino, R. Hamasuna, and T. Katoh. 2004.

Prevalence and risk factors of asymptomatic chlamydial infection among students

in Japan. Int. J. STD AIDS. 15:408-414.

90. Jo, K., Y. Ohta, J. Kitahara, T. Kodama, and E. Takahashi. 1962. Isolation of a

virus from sputum and blood of a case of psittacosis. Jpn. Med. J. 1998:31-34.

91. Jones, D. T., W. R. Taylor, and J. M. Thornton. 1992. The rapid generation of

mutation data matrices from protein sequences. CABIOS. 8:275-282.

92. Joyee, A. G., S. P. Thyagarajan, P. Rajendran, R. Hari, P. Balakrishnan, L.

Jeyaseelan, and T. Kurien. 2004. Chlamydia trachomatis genital infection in

apparently healthy adult population of Tamil Nadu, India: a population-based

study. Int. J. STD AIDS. 15:51-55.

93. Jukes, T. H., and C. R. Cantor. 1969. Evolution of protein molecules, p. 21–132. In

H. N. Munro (ed.), Mammalian protein metabolism, vol. III. Academic Press, New

York.

94. Kaleta, E. F., and E. M. Taday. 2003. Avian host range of Chlamydophila spp.

based on isolation, antigen detection and serology. Avian Pathol. 32:435–462.

95. Kaltenbock, B., N. Schmeer, and R. Schneider. 1997. Evidence for numerous omp1

alleles of porcine Chlamydia trachomatis and novel chlamydial species obtained by

PCR. J. Clin. Microbiol. 35:1835-1841.

96. Kaltenboeck, B., K. G. Kousoulas, and J. Storz. 1991. Detection and strain

differentiation of Chlamydia psittaci mediated by a two-step polymerase chain

reaction. J. Clin. Microbiol. 29:1969-1975.

126

97. Kaltenboeck, B., K. G. Kousoulas, and J. Storz. 1992. Two-step polymerase chain

reactions and restriction endonuclease analyses detect and differentiate ompA DNA

of Chlamydia spp. J. Clin. Microbiol. 30:1098-1104.

98. Kaltenboeck, B., K. G. Kousoulas, and J. Storz. 1993. Structures of and allelic

diversity and relationships among the major outer membrane protein (ompA) genes

of the four chlamydial species. J. Bacteriol. 175:487-502.

99. Kaltenboeck, B., and J. Storz. 1992. Biological properties and genetic analysis of

the ompA locus in chlamydiae isolated from swine. Am. J. Vet. Res. 53:1482-1487.

100. Kauffold, J., F. Melzer, K. Henning, K. Schulze, C. Leiding, and K. Sachse. 2006.

Prevalence of chlamydiae in boars and semen used for artificial insemination.

Theriogenology. 65:1750-1758.

101. Kazdan, J. J., J. Schachter, and M. Okumoto. 1967. Inclusion conjunctivitis in the

guinea pig. Am. J. Ophthalmol. 64:116-124.

102. Kishino, H., and M. Hasegawa. 1989. Evaluation of the maximum likelihood

estimate of the evolutionary tree topologies from DNA sequence data, and the

branching order in hominoidea. J. Mol. Evol. 29:170-179.

103. Kol, A., A. H. Lichtman, R. W. Finberg, P. Libby, and E. A. Kurt-Jones. 2000.

Cutting edge: heat shock protein (HSP) 60 activates the innate immune response:

CD14 is an essential receptor for HSP60 activation of mononuclear cells. J.

Immunol. 164:13-17.

104. Kuo, C., and W. J. Chen. 1980. A mouse model of Chlamydia trachomatis

pneumonitis. J. Infect. Dis. 141:198-202.

105. Kuo, C. C., and L. A. Campbell. 2003. Chlamydial infections of the cardiovascular

system. Front. Biosci. 8:e36-43.

106. Kuo, C. C., L. A. Jackson, L. A. Campbell, and J. T. Grayston. 1995. Chlamydia

pneumoniae (TWAR). Clin. Microbiol. Rev. 8:451-461.

127

107. Kuwabara, M., N. Tanemori, Y. Kawaguti, K. Nakamura, S. Nomiyama, M.

Terada, Y. Mitsutou, K. Imura, and M. Yamakido. 1990. Clinical features of 36

cases of psittacosis. Kansenshogaku Zasshi. 64:498-503.

108. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the

head of bacteriophage T4. Nature. 227:680-685.

109. Levison, D. A., W. Guthrie, C. Ward, D. M. Green, and P. G. Robertson. 1971.

Infective endocarditis as part of psittacosis. Lancet. 2:844-847.

110. Li, Y., M. Wexler, D. J. Richardson, P. L. Bond, and A. W. Johnston. 2005.

Screening a wide host-range, waste-water metagenomic library in tryptophan

auxotrophs of Rhizobium leguminosarum and of Escherichia coli reveals different

classes of cloned trp genes. Environ. Microbiol. 7:1927-1936.

111. Lillie, R. D. 1930. Psittacosis: rickettsia-like inclusions in man and in experimental

animals. Public Health Rep. 45:773-778.

112. Lipman, N. S., L. L. Yan, and J. C. Murphy. 1994. Probable transmission of

Chlamydia psittaci from a macaw to a cat. J. Am. Vet. Med. Assoc. 204:1479-

1480.

113. Longbottom, D., and L. J. Coulter. 2003. Animal chlamydioses and zoonotic

implications. J. Comp. Pathol. 128:217-244.

114. Lutz-Wohlgroth, L., A. Becker, E. Brugnera, Z. L. Huat, D. Zimmermann, F.

Grimm, M. Haessig, G. Greub, S. Kaps, B. Spiess, A. Pospischil, and L. Vaughan.

2006. Chlamydiales in guinea-pigs and their zoonotic potential. J. Vet .Med. A

Physiol. Pathol. Clin. Med. 53:185-193.

115. Macfarlane, J. T., and A. D. Macrae. 1983. Psittacosis. Br. Med. Bull. 39:163-167.

116. Maegawa, N., T. Emoto, H. Mori, D. Yamaguchi, T. Fujinaga, N. Tezuka, N.

Sakai, N. Ohtsuka, and T. Fukuse. 2001. Two cases of Chlamydia psittaci infection

occurring in employees of the same pet shop. Nihon Kokyuki Gakkai Zasshi.

39:753-757.

128

117. Maffei, C., A. Marracino, F. Di Stanislao, P. Pauri, M. Clementi, and P. E. Varaldo.

1987. Psittacosis in a highly endemic area in Italy. Epidemiol. Infect. 99:413-419.

118. Malaty, R., C. R. Dawson, I. Wong, C. Lyon, and J. Schachter. 1981. Serum and

tear antibodies to Chlamydia after reinfection with guinea pig inclusion

conjunctivitis agent. Invest. Ophthalmol. Vis. Sci. 21:833-841.

119. Matsumoto, A. 1988. Structural characteristics of chlamydial bodies, p. 21-45. In

A. L. Barron (ed.), Microbiology of chlamydia. CRC Press, Boca Raton, Florida.

120. Matsumoto, A., H. Bessho, R. Soejima, and J. Hino. 1984. Biological Properties of

a Chlamydia strain isolated from a pet bird, budgerigar which was kept by a

psittacosis patient. Kawasaki Med. J. 10:77-90.

121. Matsumoto, A., H. Izutsu, N. Miyashita, and M. Ohuchi. 1998. Plaque formation

by and plaque cloning of Chlamydia trachomatis biovar trachoma. J. Clin.

Microbiol. 36:3013-3019.

122. Matsumoto, A., W. Murao, H. Kumon, H. Fukushi, and R. Chahota. 2004.

Characteristics of Chlamydia sp. isolated from cervicitis. Infectious agents

surveillance report Vol. 25 No. 8 (No. 294). Infectious disease surveillance center,

National institute of infectious diseases, Japan.

123. Matsushima, H., N. Takayanagi, M. Ubukata, D. Tokunaga, S. Mori, N. Sato, K.

Kurashima, T. Yanagisawa, Y. Sugita, and M. Kanazawa. 2002. A case of

fulminant psittacosis with rhabdomyolysis. Nihon Kokyuki Gakkai Zasshi. 40:612-

616.

124. May, S. W., C. L. Kelling, M. Sabara, and J. Sandbulte. 1996. Virulence of feline

Chlamydia psittaci in mice is not a function of the major outer membrane protein

(MOMP). Vet. Microbiol. 53:355-368.

125. McClarty, G., H. Fan, and A. A. Andersen. 1993. Diversity in nucleotide

acquisition by antigenically similar Chlamydia psittaci of avian origin. FEMS

Microbiol. Lett. 108:325-331.

129

126. McClenaghan, M., A. J. Herring, and I. D. Aitken. 1984. Comparison of Chlamydia

psittaci isolates by DNA restriction endonuclease analysis. Infect. Immun. 45:384-

389.

127. Meyer, K. F. 1965. Ornithosis, p. 675-770. In H. E. Biester and L. H. Schwarte

(ed.), Diseases of poultry, 5 ed. The Iowa State University Press, Ames Iowa.

128. Miyairi, I., O. S. Mahdi, S. P. Ouellette, R. J. Belland, and G. I. Byrne. 2006.

Different growth rates of Chlamydia trachomatis biovars reflect pathotype. J.

Infect. Dis. 194:350-357.

129. Morange, A. 1895. De la psittacose, ou infection spéciale déterminée par des

perruches. PhD Thesis. Academie de Paris, France, Paris.

130. Morrison, R. P., K. Lyng, and H. D. Caldwell. 1989. Chlamydial disease

pathogenesis. Ocular hypersensitivity elicited by a genus-specific 57-kD protein. J.

Exp. Med. 169:663-675.

131. Moulder, J. W. 1991. Interaction of chlamydiae and host cells in vitro. Microbiol.

Rev. 55:143-190.

132. Moulder, J. W., T. P. Hatch, C. C. Kao, J. Schachter, and J. Storz. 1984. Genus

Chlamydia, p. 729-739. In N. R. Krieg (ed.), Bergey's manual of systematic

bacteriology, vol. 1. Baltimore: Williams and Wilkins.

133. Mueller, M., S. Postius, J. G. Thimm, K. Gueinzius, I. Muehldorfer, and C.

Hermann. 2004. Toll-like receptors 2 and 4 do not contribute to clearance of

Chlamydophila pneumoniae in mice, but are necessary for the release of

monokines. Immunobiology. 209:599-608.

134. Mukai, S., C. Mukai, K. Asaoka, and I. Suzuki. 1985. Chlamydial infection with

main symptoms of upper cervical lymphadenitis (UCLA). Isolation of chlamydiae.

Nippon Jibiinkoka Gakkai Kaiho. 88:1200-1206.

135. Murray, E. S. 1964. Guinea pig inclusion conjunctivitis virus. I. Isolation and

identification as a member of the Psittacosis-Lymphogranuloma-Trachoma group.

J. Infect. Dis. 114:1-12.

130

136. National institute of infectious diseases. 2002. Psittacosis in Japan, 1999-2002.

Infectious Agents Surveillance Report Vol. 23 No. 10 (No 272). Infectious disease

surveillance center, National institute of infectious diseases, Japan.

137. Nelson, D. E., D. P. Virok, H. Wood, C. Roshick, R. M. Johnson, W. M. Whitmire,

D. D. Crane, O. Steele-Mortimer, L. Kari, G. McClarty, and H. D. Caldwell. 2005.

Chlamydial IFN-gamma immune evasion is linked to host infection tropism. Proc.

Natl. Acad. Sci. U. S. A. 102:10658-10663.

138. Newton, P., A. Lalvani, and C. P. Conlon. 1996. Psittacosis associated with

bilateral 4th cranial nerve palsies. J. Infect. 32:63-65.

139. Okada, N., S. Murakami, R. Miwa, Y. Hara, T. Murashima, N. Ito, Y. Okazaki, T.

Yamaguchi, and K. Hirai. 1992. Occurrence of chlamydial abortion in swine. J.

Jpn. Vet. Med. Assoc. 45:655-659.

140. Olson, B. J., and W. L. Treuting. 1944. Epidemic of severe pneumonitis in bayou

region of Louisiana; epidemiological study. Public Health Rep. 59:1299-1316.

141. Page, L. A. 1958. Measurement of pathogenicity of turkey ornithosis agents for

mice. Avian Dis. 3:23-27.

142. Page, L. A. 1959. Experimental ornithosis in turkeys. Avian Dis. 3:51-66.

143. Page, L. A. 1967. Comparison of “pathotypes’’ among chlamydial (psittacosis)

strains recovered from diseased birds and mammals. Bull. Wildl. Dis. Assoc.

3:166-175.

144. Page, L. A. 1976. Observations on the involvement of wildlife in an epornitic of

chlamydiosis in domestic turkeys. J. Am. Vet. Med. Assoc. 169:932-935.

145. Page, L. A., and R. A. Bankowski. 1960. Factors affecting the production and

detection of ornithosis antibodies in infected turkeys. Am. J. Vet. Res. 21:971-978.

146. Palmer, S. R. 1982. Psittacosis in man - recent developments in the UK: a review.

J. R. Soc. Med. 75:262-267.

131

147. Pickett, M. A., J. S. Everson, and I. N. Clarke. 1988. Chlamydia psittaci ewe

abortion agent: Complete nucleotide sequence of the major outer membrane protein

gene. FEMS Microbiol. Lett. 55:229-234.

148. Poole, E., and I. Lamont. 1992. Chlamydia trachomatis serovar differentiation by

direct sequence analysis of the variable segment 4 region of the major outer

membrane protein gene. Infect. Immun. 60:1089-1094.

149. Pudjiatmoko, H. Fukushi, Y. Ochiai, T. Yamaguchi, and K. Hirai. 1997.

Phylogenetic analysis of the genus Chlamydia based on 16S rRNA gene sequences.

Int. J. Syst. Bacteriol. 47:425-431.

150. Rank, R. G., A. K. Bowlin, R. L. Reed, and T. Darville. 2003. Characterization of

chlamydial genital infection resulting from sexual transmission from male to

female guinea pigs and determination of infectious dose. Infect. Immun. 71:6148-

6154.

151. Rank, R. G., and M. M. Sanders. 1992. Pathogenesis of endometritis and salpingitis

in a guinea pig model of chlamydial genital infection. Am. J. Pathol. 140:927-936.

152. Rank, R. G., H. J. White, and A. L. Barron. 1979. Humoral immunity in the

resolution of genital infection in female guinea pigs infected with the agent of

guinea pig inclusion conjunctivitis. Infect. Immun. 26:573-579.

153. Read, T. D., G. S. Myers, R. C. Brunham, W. C. Nelson, I. T. Paulsen, J.

Heidelberg, E. Holtzapple, H. Khouri, N. B. Federova, H. A. Carty, L. A. Umayam,

D. H. Haft, J. Peterson, M. J. Beanan, O. White, S. L. Salzberg, R. C. Hsia, G.

McClarty, R. G. Rank, P. M. Bavoil, and C. M. Fraser. 2003. Genome sequence of

Chlamydophila caviae (Chlamydia psittaci GPIC): examining the role of niche-

specific genes in the evolution of the Chlamydiaceae. Nucleic Acids Res. 31:2134-

2147.

154. Reed, L. J., and H. Muench. 1938. A simple method of estimating fifty percent

endpoints. Am. J. Hyg. 27:493-497.

155. Riantawan, P., and P. Nunthapisud. 1996. Psittacosis pneumonia: a case report and

review of the literature. J. Med. Assoc. Thai. 79:55-59.

132

156. Richmond, S. J., and E. O. Caul. 1975. Fluorescent antibody studies in chlamydial

infections. J. Clin. Microbiol. 1:345-352.

157. Richmond, S. J., P. Stirling, and C. R. Ashley. 1982. Virus infecting the reticulate

bodies of an avian strain of Chlamydia psittaci. FEMS Microbiol. Lett. 14:31-36.

158. Ritter, J. 1880. Beitrag zur Frage des Pneumotyphus (Eine Hausepidemie in Uster

[Schweiz] betreffend). Deutsches Archiv für Klinische Medizin. 25:53-96.

159. Rodolakis, A., F. Bernard, and F. Lantier. 1989. Mouse models for evaluation of

virulence of Chlamydia psittaci isolated from ruminants. Res. Vet. Sci. 46:34-39.

160. Rodolakis, A., J. Salinas, and J. Papp. 1998. Recent advances on ovine chlamydial

abortion. Vet. Res. 29:275-288.

161. Rodolakis, A., and A. Souriau. 1989. Variations in the virulence of strains of

Chlamydia psittaci for pregnant ewes. Vet. Rec. 125:87-90.

162. Rodolakis, A., and A. Souriau. 1992. Restriction endonuclease analysis of DNA

from ruminant Chlamydia psittaci and its relation to mouse virulence. Vet.


163. Rogers, D. G., and A. A. Andersen. 1999. Conjunctivitis caused by a swine

Chlamydia trachomatis-like organism in gnotobiotic pigs. J. Vet. Diagn. Invest.

11:341-344.

164. Rogers, D. G., and A. A. Andersen. 2000. Intestinal lesions caused by a strain of

Chlamydia suis in weanling pigs infected at 21 days of age. J. Vet. Diagn. Invest.

12:233-239.

165. Rogers, D. G., A. A. Andersen, A. Hogg, D. L. Nielsen, and M. A. Huebert. 1993.

Conjunctivitis and keratoconjunctivitis associated with chlamydiae in swine. J. Am.

Vet. Med. Assoc. 203:1321-1323.

166. Rogers, D. G., A. A. Andersen, and B. D. Hunsaker. 1996. Lung and nasal lesions

caused by a swine chlamydial isolate in gnotobiotic pigs. J. Vet. Diagn. Invest.

8:45-55.

133

167. Sachse, K., E. Grossmann, A. Berndt, C. Schutt, K. Henning, D. Theegarten, O.

Anhenn, and P. Reinhold. 2004. Respiratory chlamydial infection based on

experimental aerosol challenge of pigs with Chlamydia suis. Comp. Immunol.

Microbiol. Infect. Dis. 27:7-23.

168. Saeki, S., I. Hirata, T. Fukusako, K. Negoro, H. Nogaki, and M. Morimatsu. 1996.

A case of psittacosis with psychiatric symptoms, abnormal EEG, and abnormal

SPECT. No To Shinkei. 48:1141-1145.

169. Saikku, P., and J. Paavonen. 1978. Single-antigen immunofluorescence test for

chlamydial antibodies. J. Clin. Microbiol. 8:119-122.

170. Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new method for

reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406-425.

171. Sambrook, J., and W. D. Russell. 2001. Molecular cloning: a laboratory manual, 3

ed, vol. 1. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

172. Samra, Z., A. Pik, A. Guidetti-Sharon, E. Yona, and Y. Weisman. 1991. Hepatitis

in a family infected by Chlamydia psittaci. J. R. Soc. Med. 84:347-348.

173. Sayada, C., A. Andersen, P. Rodriguez, F. Eb, A. Milon, J. Elion, and E. Denamur.

1994. Homogeneity of the major outer membrane protein gene of feline Chlamydia

psittaci. Res. Vet. Sci. 56:116-118.

174. Sayada, C., A. A. Andersen, C. Storey, A. Milon, F. Eb, N. Hashimoto, K. Hirai, J.

Elion, and E. Denamur. 1995. Usefulness of omp1 restriction mapping for avian

Chlamydia psittaci isolate differentiation. Res. Microbiol. 146:155-165.

175. Schachter, J. 1988. Chlamydiaceae: The chlamydiae, p. 847-890. In E. H. Lennette,

P. Halonen, and F. A. Murphy (ed.), Laboratory diagnosis of infectious diseases:

principles and practice: viral, rickettsial and chlamydial diseases., vol. 2. Springer-

Verlag, New York.

176. Schachter, J. 1990. Chlamydial infections. West. J. Med. 153:523-534.

134

177. Schachter, J. 1999. Infection and disease epidemiology, p. 139–169. In R. S.

Stephens (ed.), Chlamydia: intracellular biology, pathogenesis, and immunity.

American Society for Microbiology, Washington, DC.

178. Schiller, I., R. Koesters, R. Weilenmann, B. Kaltenboeck, and A. Pospischil. 1997.

Polymerase chain reaction (PCR) detection of porcine Chlamydia trachomatis and

ruminant Chlamydia psittaci serovar 1 DNA in formalin-fixed intestinal specimens

from swine. Zentralbl. Veterinarmed. B 44:185-191.

179. Schiller, I., R. Koesters, R. Weilenmann, R. Thoma, B. Kaltenboeck, P. Heitz, and

A. Pospischil. 1997. Mixed infections with porcine Chlamydia

trachomatis/pecorum and infections with ruminant Chlamydia psittaci serovar 1

associated with abortions in swine. Vet. Microbiol. 58:251-260.

180. Semel, J. D. 1984. Cutaneous findings in a case of psittacosis. Arch. Dermatol.

120:1227-1229.

181. Smith, K. A., K. K. Bradley, M. G. Stobierski, and L. A. Tengelsen. 2005.

Compendium of measures to control Chlamydophila psittaci (formerly Chlamydia

psittaci) infection among humans (psittacosis) and pet birds, 2005. J. Am. Vet.

Med. Assoc. 226:532-539.

182. Spalatin, J., C. E. Fraser, R. Connell, R. P. Hanson, and D. T. Berman. 1966.

Agents of psittacosis-lymphogranuloma venereum group isolated from muskrats

and snowshoe hares in Saskatchewan. Can. J. Comp. Med. Vet. Sci. 30:260-264.

183. Spears, P., and J. Storz. 1979. Biotyping of Chlamydia psittaci based on inclusion

morphology and response to diethylaminoethyl-dextran and cycloheximide. Infect.

Immun. 24:224-232.

184. Spears, P., and J. Storz. 1979. Chlamydia psittaci: growth characteristics and

enumeration of serotypes 1 and 2 in cultured cells. J. Infect. Dis. 140:959-967.

185. Stephens, R. S. 1999. Chlamydia: intracellular biology, pathology, and immunity.


135

186. Stephens, R. S., R. Sanchez-Pescador, E. A. Wagar, C. Inouye, and M. S. Urdea.

1987. Diversity of Chlamydia trachomatis major outer membrane protein genes. J.

Bacteriol. 169:3879-3885.

187. Stephens, R. S., M. R. Tam, C. C. Kuo, and R. C. Nowinski. 1982. Monoclonal

antibodies to Chlamydia trachomatis: antibody specificities and antigen

characterization. J. Immunol. 128:1083-1089.

188. Storz, J. 1971. Chlamydia and chlamydia-induced diseases. Charles C Thomas

Publisher, Limited, Springfield.

189. Stothard, D. R., G. Boguslawski, and R. B. Jones. 1998. Phylogenetic analysis of

the Chlamydia trachomatis major outer membrane protein and examination of

potential pathogenic determinants. Infect. Immun. 66:3618-3625.

190. Sturm-Ramirez, K., H. Brumblay, K. Diop, A. Gueye-Ndiaye, J. L. Sankale, I.

Thior, I. N'Doye, C. C. Hsieh, S. Mboup, and P. J. Kanki. 2000. Molecular

epidemiology of genital Chlamydia trachomatis infection in high-risk women in

Senegal, West Africa. J. Clin. Microbiol. 38:138-145.

191. Sudler, C., L. E. Hoelzle, I. Schiller, and R. K. Hoop. 2004. Molecular

characterisation of chlamydial isolates from birds. Vet. Microbiol. 98:235-241.

192. Sykes, J. E. 2005. Feline chlamydiosis. Clin. Tech. Small Anim. Pract. 20:129-134.

193. Takahashi, T., M. Masuda, T. Tsuruno, Y. Mori, I. Takashima, T. Hiramune, and

N. Kikuchi. 1997. Phylogenetic analyses of Chlamydia psittaci strains from birds

based on 16S rRNA gene sequence. J. Clin. Microbiol. 35:2908-2914.

194. Tamura, A., and N. Higashi. 1963. Purification and chemical composition of

meningopneumonitis virus. Virology. 20:596-604.

195. Taylor, H. R., S. L. Johnson, R. A. Prendergast, J. Schachter, C. R. Dawson, and A.

M. Silverstein. 1982. An animal model of trachoma II. The importance of repeated

reinfection. Invest. Ophthalmol. Vis. Sci. 23:507-515.

136

196. Teankum, K., A. Pospischil, F. Janett, E. Burgi, E. Brugnera, K. Hoelzle, A.

Polkinghorne, R. Weilenmann, D. R. Zimmermann, and N. Borel. 2006. Detection

of chlamydiae in boar semen and genital tracts. Vet. Microbiol. 116:149-157.

197. Telfer, B. L., S. A. Moberley, K. P. Hort, J. M. Branley, D. E. Dwyer, D. J.

Muscatello, P. K. Correll, J. England, and J. M. McAnulty. 2005. Probable

psittacosis outbreak linked to wild birds. Emerg. Infect. Dis. 11:391-397.

198. Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins.

1997. The CLUSTAL_X windows interface: flexible strategies for multiple

sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25:4876-

4882.

199. Thomson, N. R., C. Yeats, K. Bell, M. T. Holden, S. D. Bentley, M. Livingstone,

A. M. Cerdeno-Tarraga, B. Harris, J. Doggett, D. Ormond, K. Mungall, K. Clarke,

T. Feltwell, Z. Hance, M. Sanders, M. A. Quail, C. Price, B. G. Barrell, J. Parkhill,

and D. Longbottom. 2005. The Chlamydophila abortus genome sequence reveals

an array of variable proteins that contribute to interspecies variation. Genome Res.

15:629-640.

200. Timms, P., F. W. Eaves, A. A. Girjes, and M. F. Lavin. 1988. Comparison of

Chlamydia psittaci isolates by restriction endonuclease and DNA probe analyses.

Infect. Immun. 56:287-290.

201. Toyokawa, M., T. Kishimoto, Y. Cai, M. Ogawa, S. Shiga, I. Nishi, H. Hosotsubo,

M. Horikawa, and S. Asari. 2004. Severe Chlamydophila psittaci pneumonia

rapidly diagnosed by detection of antigen in sputum with an

immunochromatography assay. J. Infect. Chemother. 10:245-249.

202. Travnicek, M., L. Cislakova, W. Deptula, M. Stosik, and M. R. Bhide. 2002. Wild

pigeons and pheasants--a source of Chlamydophila psittaci for humans and

animals. Ann. Agric. Environ. Med. 9:253-255.

203. Treharne, J. D., and A. Shallal. 1991. The antigenic specificity of the humoral

immune response to primary and repeated ocular infections of the guinea pig with

the GPIC agent (Chlamydia psittaci). Eye. 5 (Pt 3):299-304.

137

204. van Bergen, J., H. M. Gotz, J. H. Richardus, C. J. Hoebe, J. Broer, and A. J.

Coenen. 2005. Prevalence of urogenital Chlamydia trachomatis increases

significantly with level of urbanisation and suggests targeted screening approaches:

results from the first national population based study in the Netherlands. Sex.

Transm. Infect. 81:17-23.

205. Van Loock, M., D. Vanrompay, B. Herrmann, J. Vander Stappen, G. Volckaert, B.

M. Goddeeris, and K. D. Everett. 2003. Missing links in the divergence of

Chlamydophila abortus from Chlamydophila psittaci. Int. J. Syst. Evol. Microbiol.

53:761-770.

206. Vanrompay, D., A. A. Andersen, R. Ducatelle, and F. Haesebrouck. 1993.

Serotyping of European isolates of Chlamydia psittaci from poultry and other birds.

J. Clin. Microbiol. 31:134-137.

207. Vanrompay, D., P. Butaye, C. Sayada, R. Ducatelle, and F. Haesebrouck. 1997.

Characterization of avian Chlamydia psittaci strains using omp1 restriction

mapping and serovar-specific monoclonal antibodies. Res. Microbiol. 148:327-333.

208. Vanrompay, D., E. Cox, J. Mast, B. Goddeeris, and G. Volckaert. 1998. High-level

expression of Chlamydia psittaci major outer membrane protein in COS cells and

in skeletal muscles of turkeys. Infect. Immun. 66:5494-5500.

209. Vanrompay, D., W. De Meurichy, R. Ducatelle, and F. Haesebrouck. 1994.

Pneumonia in Moorish tortoises (Testudo graeca) associated with avian serovar A

Chlamydia psittaci. Vet. Rec. 135:284-285.

210. Vanrompay, D., R. Ducatelle, and F. Haesebrouck. 1995. Chlamydia psittaci

infections: a review with emphasis on avian chlamydiosis. Vet. Microbiol. 45:93-

119.

211. Vanrompay, D., T. Geens, A. Desplanques, T. Q. Hoang, L. De Vos, M. Van

Loock, E. Huyck, C. Mirry, and E. Cox. 2004. Immunoblotting, ELISA and culture

evidence for Chlamydiaceae in sows on 258 Belgian farms. Vet. Microbiol. 99:59-

66.

138

212. Vanrompay, D., J. Mast, R. Ducatelle, F. Haesebrouck, and B. Goddeeris. 1995.

Chlamydia psittaci in turkeys: pathogenesis of infections in avian serovars A, B

and D. Vet. Microbiol. 47:245-256.

213. Verweij, P. E., J. F. Meis, R. Eijk, W. J. Melchers, and J. M. Galama. 1995. Severe

human psittacosis requiring artificial ventilation: case report and review. Clin.

Infect. Dis. 20:440-442.

214. Virok, D. P., D. E. Nelson, W. M. Whitmire, D. D. Crane, M. M. Goheen, and H.

D. Caldwell. 2005. Chlamydial infection induces pathobiotype-specific protein

tyrosine phosphorylation in epithelial cells. Infect. Immun. 73:1939-1946.

215. Volp, K., S. Mathews, P. Timms, and L. Hafner. 2001. Peptide immunization of

guinea pigs against Chlamydia psittaci (GPIC agent) infection induces good

vaginal secretion antibody response, in vitro neutralization and partial protection

against live challenge. Immunol. Cell Biol. 79:245-250.

216. Ward, M. E. 1988. The chlamydial developmental cycle, p. 71-96. In A. L. Barron

(ed.), Microbiology of chlamydia. CRC Press, Boca Raton, Florida.

217. Ward, M. E. 1999. Mechanisms of chlamydia-induced disease p. 171-210. In R. S.

Stephens (ed.), Chlamydia: intracellular biology, pathogenesis and immunity.


218. Wills, J. M., G. Watson, M. Lusher, T. S. Mair, D. Wood, and S. J. Richmond.

1990. Characterisation of Chlamydia psittaci isolated from a horse. Vet. Microbiol.

24:11-19.

219. Winsor, D. K., Jr., and J. E. Grimes. 1988. Relationship between infectivity and

cytopathology for L-929 cells, membrane proteins, and antigenicity of avian

isolates of Chlamydia psittaci. Avian Dis. 32:421-431.

220. Wong, K. H., S. K. Skelton, and H. Daugharty. 1994. Utility of complement

fixation and microimmunofluorescence assays for detecting serologic responses in

patients with clinically diagnosed psittacosis. J. Clin. Microbiol. 32:2417-2421.

139

221. Yamashita, T., and K. Hirai. 1981. Isolation of Chlamydia psittaci from imported

psittacine birds in Japan. Nippon Juigaku Zasshi. 43:561-563.

222. Yan, C., H. Fukushi, H. Matsudate, K. Ishihara, K. Yasuda, H. Kitagawa, T.

Yamaguchi, and K. Hirai. 2000. Seroepidemiological investigation of feline

chlamydiosis in cats and humans in Japan. Microbiol. Immunol. 44:155-160.

223. Yang, Z. P., C. C. Kuo, and J. T. Grayston. 1993. A mouse model of Chlamydia

pneumoniae strain TWAR pneumonitis. Infect. Immun. 61:2037-2040.

224. Yung, A. P., and M. L. Grayson. 1988. Psittacosis--a review of 135 cases. Med. J.

Aust. 148:228-233.

225. Zhang, Y. X., J. G. Fox, Y. Ho, L. Zhang, H. F. Stills, Jr., and T. F. Smith. 1993.

Comparison of the major outer-membrane protein (MOMP) gene of mouse

pneumonitis (MoPn) and hamster SFPD strains of Chlamydia trachomatis with

other Chlamydia strains. Mol. Biol. Evol. 10:1327-1342.

226. Zhang, Y. X., S. G. Morrison, H. D. Caldwell, and W. Baehr. 1989. Cloning and

sequence analysis of the major outer membrane protein genes of two Chlamydia

psittaci strains. Infect. Immun. 57:1621-1625.

227. Zhao, Q., J. Schachter, and R. S. Stephens. 1993. Lack of allelic polymorphism for

the major outer membrane protein gene of the agent of guinea pig inclusion

conjunctivitis (Chlamydia psittaci). Infect. Immun. 61:3078-3080.

140

First of all, I wish to express my sincere gratitude to my major academic supervisor,

Prof. Hideto Fukushi, Laboratory of Veterinary Microbiology, Faculty of Applied

Biological Sciences, Gifu University, for his solicitous and intellectual guidance during this

study. I am also deeply indebted to my co-supervisors, Dr. Tsuyoshi Yamaguchi,

Laboratory of Veterinary Microbiology, Gifu University and Prof. Shinagawa Kunihiro,

Department of Veterinary Microbiology, Faculty of Agriculture, Iwate University for their

pragmatic suggestions and supervision and also reviewing the manuscript.

I am also expressing my heartfelt thanks to Prof. Akira Matsumoto, Kawasaki

Medical School for his generosity in providing valuable biological materials for this study.

I am also thankfully acknowledging the treasured academic impetus provided by Prof.

Katsuya Hirai (retired) and Dr. Kenji Ohya, Laboratory of Veterinary Microbiology, Gifu

University during my stay in this laboratory.

I am sincerely grateful to Prof. Hishashi Inokuma, Obihiro University of

Agriculture, Prof. Eiichi Honda, Tokyo University of Agriculture and Technology and Prof.

Sekizaki Tsutomu, National Institute of Animal Health for their critical reviewing and

valuable suggestions during the preparation of this thesis.

I am also earnestly thankful to Dr. Karin D. Everett for her precious thoughts and

kind help in the analysis of genetic data during this study. I would also like to thank all the

veterinarians and field stall of different veterinary hospitals, bird parks, and zoos for their

kind help in sample collection.

I am also truly appreciative and very much grateful to all my fellow laboratory

students especially Dr. Pagamjav Ochir, Dr. Ando Masako, Dr. Yan Cai, Dr. Min Thin

Maw, Dr. Hirohito Ogawa and Dr. Souichi Yamada for their benevolent help in academic

and non-academic matters. Special thanks are also due to the non-academic staff of this

ACKNOWLEGDMENTS

141

laboratory and the office of the United Graduate School of Veterinary Sciences for their

generous help.

I will always be indebted to Prof. R. C. Katoch and Dr. Madeep Sharma and all my

teachers and friends in India for making me worth to reach at this academic echelon.

I also express my sincere thanks to the Ministry of Education, Culture, Sports and

Science of Japan for the scholarship and financial support to my research in Japan. I am

also grateful to the authorities of CSK HP Agriculture University, Palampur for granting

me study leave and the Ministry of Human Resource Development, Government of India

for nominating me for this study.

The words cannot express the enduring and compassionate support of my wife

Praveen during some topsy-turvy moments in this period. I may not be able to compensate

the time that the twinkling little eyes of my son Shashwat have spent in waiting for his

father at home. The affectionate and ever reliable personal support of my brother Dr.

Rakesh Kumar Chahota and his family and Ashwani is cherished from heart.

Finally, I dedicate this thesis to my parents Sh. Jagdish Ram and Smt. Pushpa Rani

for their ever-lasting blessings that will always inspire me along with HIS ALMIGHTY.

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Title Pathogenesis of Chlamydial Infections( 本文（FULLTEXT ...repository.lib.gifu-u.ac.jp/bitstream/20.500.12099/21409/...conjunctivitis in cats, particularly kittens (192). Studies